Pyrrolobenzodiazepine conjugates

ABSTRACT

A compound of formula I and salts and solvates thereof, wherein: R 6  and R 9  are independently selected from H, R, OH, OR, SH, SR, NH 2 , NHR, NRR′, nitro, Me 3 Sn and halo; where R and R′ are independently selected from optionally substituted C 1-12  alkyl, C 3-20  heterocyclyl and C 5-20  aryl groups; R 7  is selected from H, R, OH, OR, SH, SR, NH 2 , NHR, NRR′, nitro, Me 3 Sn and halo; R″ is a C 3-12  alkylene group, which chain may be interrupted by one or more heteroatoms, e.g. O, S, NR N2  (where R N2  is H or C 1-4  alkyl), and/or aromatic rings, e.g. benzene or pyridine; Y and Y′ are selected from O, S, or NH; R 6 , R 7 , R 9  are selected from the same groups as R 6 , R 7  and R 9  respectively; R 11b  is selected from OH, OR A , where R A  is C 1-4  alkyl; and R L  is a linker for connection to a cell binding agent.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a national phase application under 35 U.S.C. § 371of International Application No. PCT/EP2018/059846, filed Apr. 18, 2018,which claims the benefit of Great Britain Application No. 1721337.2,filed Dec. 19, 2017, and Great Britain Application No. 1706133.4, filedApr. 18, 2017, each of which is herein incorporated by reference.

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

Incorporated by reference in its entirety herein is a computer-readablenucleotide/amino acid sequence listing submitted concurrently herewithand identified as follows: One 47,814 Byte ASCII (Text) file named“2020-01-02_38075-251_SQL_ST25,” created on Jan. 2, 2020.

The present invention relates to conjugates comprisingpyrrolobenzodiazepines and related dimers (PBDs), and the precursor druglinkers used to make such conjugates.

BACKGROUND TO THE INVENTION

Some pyrrolobenzodiazepines (PBDs) have the ability to recognise andbond to specific sequences of DNA; the preferred sequence is PuGPu. Thefirst PBD antitumour antibiotic, anthramycin, was discovered in 1965(Leimgruber, et al., J. Am. Chem. Soc., 87, 5793-5795 (1965);Leimgruber, et al., J. Am. Chem. Soc., 87, 5791-5793 (1965)). Sincethen, a number of naturally occurring PBDs have been reported, and over10 synthetic routes have been developed to a variety of analogues(Thurston, et al., Chem. Rev. 1994, 433-465 (1994)). Family membersinclude abbeymycin (Hochlowski, et al., J. Antibiotics, 40, 145-148(1987)), chicamycin (Konishi, et al., J. Antibiotics, 37, 200-206(1984)), DC-81 (Japanese Patent 58-180 487; Thurston, et al., Chem.Brit., 26, 767-772 (1990); Bose, et al., Tetrahedron, 48, 751-758(1992)), mazethramycin (Kuminoto, et al., J. Antibiotics, 33, 665-667(1980)), neothramycins A and B (Takeuchi, et al., J. Antibiotics, 29,93-96 (1976)), porothramycin (Tsunakawa, et al., J. Antibiotics, 41,1366-1373 (1988)), prothracarcin (Shimizu, et al, J. Antibiotics, 29,2492-2503 (1982); Langley and Thurston, J. Org. Chem., 52, 91-97(1987)), sibanomicin (DC-102)(Hara, et al., J. Antibiotics, 41, 702-704(1988); Itoh, et al., J. Antibiotics, 41, 1281-1284 (1988)), sibiromycin(Leber, et al., J. Am. Chem. Soc., 110, 2992-2993 (1988)) and tomamycin(Arima, et al., J. Antibiotics, 25, 437-444 (1972)). PBDs are of thegeneral structure:

They differ in the number, type and position of substituents, in boththeir aromatic A rings and pyrrolo C rings, and in the degree ofsaturation of the C ring. In the B-ring there is either an imine (N═C),a carbinolamine (NH—CH(OH)), or a carbinolamine methyl ether(NH—CH(OMe))ram at the N10-C11 position which is the electrophiliccentre responsible for alkylating DNA. All of the known natural productshave an (S)-configuration at the chiral C11a position which providesthem with a right-handed twist when viewed from the C ring towards the Aring. This gives them the appropriate three-dimensional shape forisohelicity with the minor groove of B-form DNA, leading to a snug fitat the binding site (Kohn, In Antibiotics III. Springer-Verlag, NewYork, pp. 3-11 (1975); Hurley and Needham-VanDevanter, Acc. Chem. Res.,19, 230-237 (1986)). Their ability to form an adduct in the minorgroove, enables them to interfere with DNA processing, hence their useas antitumour agents.

It has been previously disclosed that the biological activity of thismolecules can be potentiated by joining two PBD units together throughtheir C8/C′-hydroxyl functionalities via a flexible alkylene linker(Bose, D. S., et al., J. Am. Chem. Soc., 114, 4939-4941 (1992);Thurston, D. E., et al., J. Org. Chem., 61, 8141-8147 (1996)). The PBDdimers are thought to form sequence-selective DNA lesions such as thepalindromic 5′-Pu-GATC-Py-3′ interstrand cross-link (Smellie, M., etal., Biochemistry, 42, 8232-8239 (2003); Martin, C., et al.,Biochemistry, 44, 4135-4147) which is thought to be mainly responsiblefor their biological activity.

The first dimers (Bose, D. S., et al., J. Am. Chem. Soc., 114, 4939-4941(1992)) were of the general formula:

where n is from 3 to 6. The compounds where n were 3 and 5 showedpromising cytoxicity in vitro. However when antitumor activity of thethe n=3 compound (DSB-120) was studied (Walton, M., et al., CancerChemother Pharmacol (1996) 38: 431. doi:10.1007/s002800050507), this wasfound not to be as promising. This lack of promise was thought to be “aconsequence of low tumour selectivity and drug uptake as a result ofhigh protein binding and/or extensive drug metabolism in vivo”.

In order to improve on these compounds, compounds were investigated(Gregson, S. J., et al., Chem. Commun., 1999, 797-798. doi:10.1039/A809791G) with the “inclusion of C2/C2′ substituents that shouldfollow the contour of the host minor groove”. This compound SG2000(SJG-136):

was found to have “exquisite cytotoxicity in the picomolar region . . .some 9000-fold more potent that DSB-120.”

This compound (also discussed in Gregson, S., et al., J. Med. Chem., 44,737-748 (2001); Alley, M. C., et al., Cancer Research, 64, 6700-6706(2004); and Hartley, J. A., et al., Cancer Research, 64, 6693-6699(2004)) has been involved in clinical trials as a standalone agent, forexample, NCT02034227 investigating its use in treating Acute MyeloidLeukemia and Chronic Lymphocytic Leukemia (see:https://www.clinicaltrials.gov/ct2/show/NCT02034227).

Dimeric PBD compounds bearing C2 aryl substituents alongsideendo-unsaturation, such as SG2202 (ZC-207), are disclosed in WO2005/085251:

and in WO2006/111759, bisulphites of such PBD compounds, for exampleSG2285 (ZC-423):

These compounds have been shown to be highly useful cytotoxic agents(Howard, P. W., et al., Bioorg. Med. Chem. (2009), doi:10.1016/j.bmcl.2009.09.012).

In a review of PBD containing ADCs (Mantaj, J., et al., Angew. Chem.Int. Ed. (2016), 55, 2-29; DOI: 10.1002/anie.201510610), the SAR of PBDdimers is discussed. The summary of the SAR is presented in FIG. 3-B“C2-exo and C1-C2/C2-C3 unsaturation enhances activity”. A more detaileddiscussion is found at section 2.4 which says:

“DSB-120 has poor activity in vivo, attributed partly to its highreactivity with cellular thiol-containing molecules such as glutathione.However, introduction of C2/C2′-exo unsaturation as in SJG-136 led to anoverall increase in DNA-binding affinity and cytotoxicity, and a lowerreactivity toward cellular nucleophiles with more of the agentpotentially reaching its target DNA.”

WO 2007/085930 describes the preparation of dimer PBD compounds havinglinker groups for connection to a cell binding agent, such as anantibody. The linker is present in the bridge linking the monomer PBDunits of the dimer.

Dimer PBD compounds having linker groups for connection to a cellbinding agent, such as an antibody, are described in WO 2011/130598. Thelinker in these compounds is attached to one of the available N10positions, and are generally cleaved by action of an enzyme on thelinker group. The dimer PBD compounds have either endo or exounsaturation in the C-ring.

WO 2014/057074 and WO 2015/052322 describes specific PBD dimerconjugates bound via the N10 position on one monomer, and all thesecompounds have endo unsaturation in the C-ring.

WO2014/096365 discloses the compound:

where the lack of unsaturation in the C-ring is coupled with the B-ringbeing a dilactam and therefore not having the ability to covalently bindDNA.

DISCLOSURE OF THE INVENTION

The present invention provides PBD dimer drug linkers and conjugateswhere neither C-ring has endo- or exo-unsaturation.

A first aspect of the present invention comprises a compound with theformula I:

and salts and solvates thereof, wherein:R⁶ and R⁹ are independently selected from H, R, OH, OR, SH, SR, NH₂,NHR, NRR′, nitro, Me₃Sn and halo;where R and R′ are independently selected from optionally substitutedC₁₋₁₂ alkyl, C₃₋₂₀ heterocyclyl and C₅₋₂₀ aryl groups;R⁷ is selected from H, R, OH, OR, SH, SR, NH₂, NHR, NRR′, nitro, Me₃Snand halo;R″ is a C₃₋₁₂ alkylene group, which chain may be interrupted by one ormore heteroatoms, e.g. O, S, NR^(N2) (where R^(N2) is H or C₁₋₄ alkyl),and/or aromatic rings, e.g. benzene or pyridine;Y and Y′ are selected from O, S, or NH;R^(6′), R^(7′), R^(9′) are selected from the same groups as R⁶, R⁷ andR⁹ respectively;R^(11b) is selected from OH, OR^(A), where R^(A) is C₁₋₄ alkyl; andR^(L) is a linker for connection to a cell binding agent, which isselected from:(iiia):

wherein

Q is:

where Q^(X) is such that Q is an amino-acid residue, a dipeptide residueor a tripeptide residue;

X is:

where a=0 to 5, b=0 to 16, c=0 or 1, d=0 to 5;G^(L) is a linker for connecting to a Ligand Unit; and(iiib):

where R^(L1) and R^(L2) are independently selected from H and methyl, ortogether with the carbon atom to which they are bound form acyclopropylene or cyclobutylene group;and e is 0 or 1;either

-   -   (a) R²⁰ is H, and R²¹ is OH or OR^(A), where R^(A) is C₁₋₄        alkyl; or    -   (b) R²⁰ and R²¹ form a nitrogen-carbon double bond between the        nitrogen and carbon atoms to which they are bound; or    -   (c) R²⁰ is H and R²¹ is SO_(z)M, where z is 2 or 3 and M is a        monovalent pharmaceutically acceptable cation; or    -   (d) R²⁰ is H and R²¹ is H; or    -   (e) R²¹ is OH or OR^(A), where R^(A) is C₁₋₄ alkyl and R²⁰ is        selected from:        -   (d-i)

-   -   -   (d-ii)

-   -   -   (d-iii)

-   -   -    where R^(Z) is selected from:            -   (z-i)

-   -   -   -   (z-ii) OC(═O)CH₃;            -   (z-iii) NO₂;            -   (z-iv) OMe;            -   (z-v) glucoronide;            -   (z-vi) —C(═O)—X₁—NHC(═O)X₂—NH—R^(ZC), where                —C(═O)—X₁—NH— and —C(═O)—X₂—NH— represent natural amino                acid residues and R^(ZC) is selected from Me, OMe,                OCH₂CH₂OMe.

Such drug linkers have been found to undergo ready conjugation to ligandunits such as antibodies.

A second aspect of the present invention provides Conjugates of formulaII:

L-(D^(L))_(p)  (II)

wherein L is a Ligand unit (i.e., a targeting agent), D^(L) is a DrugLinker unit of formula I′:

wherein R⁶, R⁷, R⁹, R^(11b), Y, R″, Y′, R^(6′), R^(7′), R^(9′), R²⁰ andR²¹ are as defined in the first aspect of the invention;R^(LL) is a linker for connection to a cell binding agent, which isselected from:(iiia):

where Q and X are as defined in the first aspect and G^(LL) is a linkerconnected to a Ligand Unit; and(iiib):

where R^(L1) and R^(L2) are as defined in the first aspect;wherein p is an integer of from 1 to 20.

The Ligand unit, described more fully below, is a targeting agent thatbinds to a target moiety. The Ligand unit can, for example, specificallybind to a cell component (a Cell Binding Agent) or to other targetmolecules of interest. The Ligand unit can be, for example, a protein,polypeptide or peptide, such as an antibody, an antigen-binding fragmentof an antibody, or other binding agent, such as an Fc fusion protein.

These conjugates have been found to possess a high tolerability whichleads to a high therapeutic index, thus making them promising candidatesfor clinical development.

A third aspect of the present invention provides the use of a conjugateof the second aspect of the invention in the manufacture of a medicamentfor treating a proliferative disease. The third aspect also provides aconjugate of the second aspect of the invention for use in the treatmentof a proliferative disease. The third aspect also provides a method oftreating a proliferative disease comprising administering atherapeutically effective amount of a conjugate of the second aspect ofthe invention to a patient in need thereof.

One of ordinary skill in the art is readily able to determine whether ornot a candidate conjugate treats a proliferative condition for anyparticular cell type. For example, assays which may conveniently be usedto assess the activity offered by a particular compound are described inthe examples below.

A fourth aspect of the present invention provides the synthesis of aconjugate of the second aspect of the invention comprising conjugating acompound (drug linker) of the first aspect of the invention with aLigand Unit.

Definitions Substituents

The phrase “optionally substituted” as used herein, pertains to a parentgroup which may be unsubstituted or which may be substituted.

Unless otherwise specified, the term “substituted” as used herein,pertains to a parent group which bears one or more substituents. Theterm “substituent” is used herein in the conventional sense and refersto a chemical moiety which is covalently attached to, or if appropriate,fused to, a parent group. A wide variety of substituents are well known,and methods for their formation and introduction into a variety ofparent groups are also well known.

Examples of substituents are described in more detail below.

C₁₋₁₂ alkyl: The term “C₁₋₁₂ alkyl” as used herein, pertains to amonovalent moiety obtained by removing a hydrogen atom from a carbonatom of a hydrocarbon compound having from 1 to 12 carbon atoms, whichmay be aliphatic or alicyclic, and which may be saturated or unsaturated(e.g. partially unsaturated, fully unsaturated). The term “C₁₋₄ alkyl”as used herein, pertains to a monovalent moiety obtained by removing ahydrogen atom from a carbon atom of a hydrocarbon compound having from 1to 4 carbon atoms, which may be aliphatic or alicyclic, and which may besaturated or unsaturated (e.g. partially unsaturated, fullyunsaturated). Thus, the term “alkyl” includes the sub-classes alkenyl,alkynyl, cycloalkyl, etc., discussed below.

Examples of saturated alkyl groups include, but are not limited to,methyl (C₁), ethyl (C₂), propyl (C₃), butyl (C₄), pentyl (C₅), hexyl(C₆) and heptyl (C₇).

Examples of saturated linear alkyl groups include, but are not limitedto, methyl (C₁), ethyl (C₂), n-propyl (C₃), n-butyl (C₄), n-pentyl(amyl) (C₅), n-hexyl (C₆) and n-heptyl (C₇).

Examples of saturated branched alkyl groups include iso-propyl (C₃),iso-butyl (C₄), sec-butyl (C₄), tert-butyl (C₄), iso-pentyl (C₅), andneo-pentyl (C₅).

C₂₋₁₂ Alkenyl: The term “C₂₋₁₂ alkenyl” as used herein, pertains to analkyl group having one or more carbon-carbon double bonds.

Examples of unsaturated alkenyl groups include, but are not limited to,ethenyl (vinyl, —CH═CH₂), 1-propenyl (—CH═CH—CH₃), 2-propenyl (allyl,—CH—CH═CH₂), isopropenyl (1-methylvinyl, —C(CH₃)═CH₂), butenyl (C₄),pentenyl (C₅), and hexenyl (C₆).

C₂₋₁₂ alkynyl: The term “C₂₋₁₂ alkynyl” as used herein, pertains to analkyl group having one or more carbon-carbon triple bonds.

Examples of unsaturated alkynyl groups include, but are not limited to,ethynyl (—C≡CH) and 2-propynyl (propargyl, —CH₂—C≡CH).

C₃₋₁₂ cycloalkyl: The term “C₃₋₁₂ cycloalkyl” as used herein, pertainsto an alkyl group which is also a cyclyl group; that is, a monovalentmoiety obtained by removing a hydrogen atom from an alicyclic ring atomof a cyclic hydrocarbon (carbocyclic) compound, which moiety has from 3to 7 carbon atoms, including from 3 to 7 ring atoms.

Examples of cycloalkyl groups include, but are not limited to, thosederived from:

-   -   saturated monocyclic hydrocarbon compounds:        cyclopropane (C₃), cyclobutane (C₄), cyclopentane (C₅),        cyclohexane (C₆), cycloheptane (C₇), methylcyclopropane (C₄),        dimethylcyclopropane (C₅), methylcyclobutane (C₅),        dimethylcyclobutane (C₆), methylcyclopentane (C₆),        dimethylcyclopentane (C₇) and methylcyclohexane (C₇);    -   unsaturated monocyclic hydrocarbon compounds:        cyclopropene (C₃), cyclobutene (C₄), cyclopentene (C₅),        cyclohexene (C₆), methylcyclopropene (C₄), dimethylcyclopropene        (C₅), methylcyclobutene (C₅), dimethylcyclobutene (C₆),        methylcyclopentene (C₆), dimethylcyclopentene (C₇) and        methylcyclohexene (C₇); and    -   saturated polycyclic hydrocarbon compounds:        norcarane (C₇), norpinane (C₇), norbornane (C₇).

C₃₋₂₀ heterocyclyl: The term “C₃₋₂₀ heterocyclyl” as used herein,pertains to a monovalent moiety obtained by removing a hydrogen atomfrom a ring atom of a heterocyclic compound, which moiety has from 3 to20 ring atoms, of which from 1 to 10 are ring heteroatoms. Preferably,each ring has from 3 to 7 ring atoms, of which from 1 to 4 are ringheteroatoms.

In this context, the prefixes (e.g. C₃₋₂₀, C₃₋₇, C₅₋₆, etc.) denote thenumber of ring atoms, or range of number of ring atoms, whether carbonatoms or heteroatoms. For example, the term “C₅₋₆heterocyclyl”, as usedherein, pertains to a heterocyclyl group having 5 or 6 ring atoms.

Examples of monocyclic heterocyclyl groups include, but are not limitedto, those derived from:

N₁: aziridine (C₃), azetidine (C₄), pyrrolidine (tetrahydropyrrole)(C₅), pyrroline (e.g., 3-pyrroline, 2,5-dihydropyrrole) (C₅), 2H-pyrroleor 3H-pyrrole (isopyrrole, isoazole) (C₅), piperidine (C₆),dihydropyridine (C₆), tetrahydropyridine (C₆), azepine (C₇);O₁: oxirane (C₃), oxetane (C₄), oxolane (tetrahydrofuran) (C₅), oxole(dihydrofuran) (C₅), oxane (tetrahydropyran) (C₆), dihydropyran (C₆),pyran (C₆), oxepin (C₇);S₁: thiirane (C₃), thietane (C₄), thiolane (tetrahydrothiophene) (C₅),thiane (tetrahydrothiopyran) (C₆), thiepane (C₇);O₂: dioxolane (C₅), dioxane (C₆), and dioxepane (C₇);O₃: trioxane (C₆);N₂: imidazolidine (C₅), pyrazolidine (diazolidine) (C₅), imidazoline(C₅), pyrazoline (dihydropyrazole) (C₅), piperazine (C₆);N₁O₁: tetrahydrooxazole (C₅), dihydrooxazole (C₅), tetrahydroisoxazole(C₅), dihydroisoxazole (C₅), morpholine (C₆), tetrahydrooxazine (C₆),dihydrooxazine (C₆), oxazine (C₆);N₁S1: thiazoline (C₅), thiazolidine (C₅), thiomorpholine (C₆);N₂O₁: oxadiazine (C₆);O₁S₁: oxathiole (C₅) and oxathiane (thioxane) (C₆); and,N₁O₁S₁: oxathiazine (C₆).

Examples of substituted monocyclic heterocyclyl groups include thosederived from saccharides, in cyclic form, for example, furanoses (C₅),such as arabinofuranose, lyxofuranose, ribofuranose, and xylofuranse,and pyranoses (C₆), such as allopyranose, altropyranose, glucopyranose,mannopyranose, gulopyranose, idopyranose, galactopyranose, andtalopyranose.

C₅₋₂₀ aryl: The term “C₅₋₂₀ aryl”, as used herein, pertains to amonovalent moiety obtained by removing a hydrogen atom from an aromaticring atom of an aromatic compound, which moiety has from 3 to 20 ringatoms. The term “C₅₋₇ aryl”, as used herein, pertains to a monovalentmoiety obtained by removing a hydrogen atom from an aromatic ring atomof an aromatic compound, which moiety has from 5 to 7 ring atoms and theterm “C₅₋₁₀ aryl”, as used herein, pertains to a monovalent moietyobtained by removing a hydrogen atom from an aromatic ring atom of anaromatic compound, which moiety has from 5 to 10 ring atoms. Preferably,each ring has from 5 to 7 ring atoms.

In this context, the prefixes (e.g. C₃₋₂₀, C₅₋₇, C₅₋₆, C₅₋₁₀, etc.)denote the number of ring atoms, or range of number of ring atoms,whether carbon atoms or heteroatoms. For example, the term “C₅₋₆ aryl”as used herein, pertains to an aryl group having 5 or 6 ring atoms.

The ring atoms may be all carbon atoms, as in “carboaryl groups”.

Examples of carboaryl groups include, but are not limited to, thosederived from benzene (i.e. phenyl) (C₆), naphthalene (C₁₀), azulene(C₁₀), anthracene (C₁₄), phenanthrene (C₁₄), naphthacene (C₁₈), andpyrene (C₁₆).

Examples of aryl groups which comprise fused rings, at least one ofwhich is an aromatic ring, include, but are not limited to, groupsderived from indane (e.g. 2,3-dihydro-1H-indene) (C₅), indene (C₅),isoindene (C₅), tetraline (1,2,3,4-tetrahydronaphthalene (C₁₀),acenaphthene (C₁₂), fluorene (C₁₃), phenalene (C₁₃), acephenanthrene(C₁₅), and aceanthrene (C₁₆).

Alternatively, the ring atoms may include one or more heteroatoms, as in“heteroaryl groups”. Examples of monocyclic heteroaryl groups include,but are not limited to, those derived from:

N₁: pyrrole (azole) (C₅), pyridine (azine) (C₆);O₁: furan (oxole) (C₅);S₁: thiophene (thiole) (C₅);N₁O₁: oxazole (C₅), isoxazole (C₅), isoxazine (C₆);N₂O₁: oxadiazole (furazan) (C₅);N₃O₁: oxatriazole (C₅);N₁S₁: thiazole (C₅), isothiazole (C₅);N₂: imidazole (1,3-diazole) (C₅), pyrazole (1,2-diazole) (C₅),pyridazine (1,2-diazine) (C₆), pyrimidine (1,3-diazine) (C₆) (e.g.,cytosine, thymine, uracil), pyrazine (1,4-diazine) (C₆);N₃: triazole (C₅), triazine (C₆); and,N₄: tetrazole (C₅).

Examples of heteroaryl which comprise fused rings, include, but are notlimited to:

-   -   C₉ (with 2 fused rings) derived from benzofuran (O₁),        isobenzofuran (O₁), indole (N₁), isoindole (N₁), indolizine        (N₁), indoline (N₁), isoindoline (N₁), purine (N₄) (e.g.,        adenine, guanine), benzimidazole (N₂), indazole (N₂),        benzoxazole (N₁O₁), benzisoxazole (N₁O₁), benzodioxole (O₂),        benzofurazan (N₂O₁), benzotriazole (N₃), benzothiofuran (S₁),        benzothiazole (N₁S₁), benzothiadiazole (N₂S);    -   C₁₀ (with 2 fused rings) derived from chromene (O₁), isochromene        (O₁), chroman (O₁), isochroman (O₁), benzodioxan (O₂), quinoline        (N₁), isoquinoline (N₁), quinolizine (N₁), benzoxazine (N₁O₁),        benzodiazine (N₂), pyridopyridine (N₂), quinoxaline (N₂),        quinazoline (N₂), cinnoline (N₂), phthalazine (N₂),        naphthyridine (N₂), pteridine (N₄);    -   C₁₁ (with 2 fused rings) derived from benzodiazepine (N₂);    -   C₁₃ (with 3 fused rings) derived from carbazole (N₁),        dibenzofuran (O₁), dibenzothiophene (S₁), carboline (N₂),        perimidine (N₂), pyridoindole (N₂); and,    -   C₁₄ (with 3 fused rings) derived from acridine (N₁), xanthene        (O₁), thioxanthene (S₁), oxanthrene (O₂), phenoxathiin (O₁S₁),        phenazine (N₂), phenoxazine (N₁O₁), phenothiazine (N₁S₁),        thianthrene (S₂), phenanthridine (N₁), phenanthroline (N₂),        phenazine (N₂).

The above groups, whether alone or part of another substituent, maythemselves optionally be substituted with one or more groups selectedfrom themselves and the additional substituents listed below.

Halo: —F, —Cl, —Br, and —I.

Hydroxy: —OH.

Ether: —OR, wherein R is an ether substituent, for example, a C₁₋₇ alkylgroup (also referred to as a C₁₋₇ alkoxy group, discussed below), aC₃₋₂₀ heterocyclyl group (also referred to as a C₃₋₂₀ heterocyclyloxygroup), or a C₅₋₂₀ aryl group (also referred to as a C₅₋₂₀ aryloxygroup), preferably a C₀₋₇alkyl group.

Alkoxy: —OR, wherein R is an alkyl group, for example, a C₁₋₇ alkylgroup. Examples of C₁₋₇ alkoxy groups include, but are not limited to,—OMe (methoxy), —OEt (ethoxy), —O(nPr) (n-propoxy), —O(iPr)(isopropoxy), —O(nBu) (n-butoxy), —O(sBu) (sec-butoxy), —O(iBu)(isobutoxy), and —O(tBu) (tert-butoxy).

Acetal: —CH(OR¹)(OR²), wherein R¹ and R² are independently acetalsubstituents, for example, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclylgroup, or a C₅₋₂₀ aryl group, preferably a C₁₋₇ alkyl group, or, in thecase of a “cyclic” acetal group, R¹ and R², taken together with the twooxygen atoms to which they are attached, and the carbon atoms to whichthey are attached, form a heterocyclic ring having from 4 to 8 ringatoms. Examples of acetal groups include, but are not limited to,—CH(OMe)₂, —CH(OEt)₂, and —CH(OMe)(OEt).

Hemiacetal: —CH(OH)(OR¹), wherein R¹ is a hemiacetal substituent, forexample, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ arylgroup, preferably a C₁₋₇ alkyl group.

Examples of hemiacetal groups include, but are not limited to,—CH(OH)(OMe) and —CH(OH)(OEt).

Ketal: —CR(OR¹)(OR²), where R¹ and R² are as defined for acetals, and Ris a ketal substituent other than hydrogen, for example, a C₁₋₇ alkylgroup, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group, preferably aC₁₋₇ alkyl group. Examples ketal groups include, but are not limited to,—C(Me)(OMe)₂, —C(Me)(OEt)₂, —C(Me)(OMe)(OEt), —C(Et)(OMe)₂,—C(Et)(OEt)₂, and —C(Et)(OMe)(OEt).

Hemiketal: —CR(OH)(OR¹), where R¹ is as defined for hemiacetals, and Ris a hemiketal substituent other than hydrogen, for example, a C₁₋₇alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group,preferably a C₁₋₇ alkyl group. Examples of hemiacetal groups include,but are not limited to, —C(Me)(OH)(OMe), —C(Et)(OH)(OMe),—C(Me)(OH)(OEt), and —C(Et)(OH)(OEt).

Oxo (keto, -one): ═O.

Thione (thioketone): ═S.

Imino (imine): ═NR, wherein R is an imino substituent, for example,hydrogen, C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ arylgroup, preferably hydrogen or a C₁₋₇ alkyl group. Examples of estergroups include, but are not limited to, ═NH, ═NMe, =NEt, and ═NPh.

Formyl (carbaldehyde, carboxaldehyde): —C(═O)H.

Acyl (keto): —C(═O)R, wherein R is an acyl substituent, for example, aC₁₋₇ alkyl group (also referred to as C₁₋₇ alkylacyl or C₁₋₇ alkanoyl),a C₃₋₂₀ heterocyclyl group (also referred to as C₃₋₂₀ heterocyclylacyl),or a C₅₋₂₀ aryl group (also referred to as C₅₋₂₀ arylacyl), preferably aC₁₋₇ alkyl group. Examples of acyl groups include, but are not limitedto, —C(═O)CH₃ (acetyl), —C(═O)CH₂CH₃ (propionyl), —C(═O)C(CH₃)₃(t-butyryl), and —C(═O)Ph (benzoyl, phenone).

Carboxy (carboxylic acid): —C(═O)OH.

Thiocarboxy (thiocarboxylic acid): —C(═S)SH.

Thiolocarboxy (thiolocarboxylic acid): —C(═O)SH.

Thionocarboxy (thionocarboxylic acid): —C(═S)OH.

Imidic acid: —C(═NH)OH.

Hydroxamic acid: —C(═NOH)OH.

Ester (carboxylate, carboxylic acid ester, oxycarbonyl): —C(═O)OR,wherein R is an ester substituent, for example, a C₁₋₇ alkyl group, aC₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group, preferably a C₁₋₇ alkylgroup. Examples of ester groups include, but are not limited to,—C(═O)OCH₃, —C(═O)OCH₂CH₃, —C(═O)OC(CH₃)₃, and —C(═O)OPh.

Acyloxy (reverse ester): —OC(═O)R, wherein R is an acyloxy substituent,for example, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀aryl group, preferably a C₁₋₇ alkyl group.

Examples of acyloxy groups include, but are not limited to, —OC(═O)CH₃(acetoxy), —OC(═O)CH₂CH₃, —OC(═O)C(CH₃)₃, —OC(═O)Ph, and —OC(═O)CH₂Ph.

Oxycarboyloxy: —OC(═O)OR, wherein R is an ester substituent, forexample, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ arylgroup, preferably a C₁₋₇ alkyl group. Examples of ester groups include,but are not limited to, —OC(═O)OCH₃, —OC(═O)OCH₂CH₃, —OC(═O)OC(CH₃)₃,and —OC(═O)OPh.

Amino: —NR¹R², wherein R¹ and R² are independently amino substituents,for example, hydrogen, a C₁₋₇ alkyl group (also referred to as C₁₋₇alkylamino or di-C₁₋₇ alkylamino), a C₃₋₂₀ heterocyclyl group, or aC₅₋₂₀ aryl group, preferably H or a C₁₋₇ alkyl group, or, in the case ofa “cyclic” amino group, R¹ and R², taken together with the nitrogen atomto which they are attached, form a heterocyclic ring having from 4 to 8ring atoms. Amino groups may be primary (—NH₂), secondary (—NHR¹), ortertiary (—NHR¹R²), and in cationic form, may be quaternary (—⁺NR¹R²R³).Examples of amino groups include, but are not limited to, —NH₂, —NHCH₃,—NHC(CH₃)₂, —N(CH₃)₂, —N(CH₂CH₃)₂, and —NHPh. Examples of cyclic aminogroups include, but are not limited to, aziridino, azetidino,pyrrolidino, piperidino, piperazino, morpholino, and thiomorpholino.

Amido (carbamoyl, carbamyl, aminocarbonyl, carboxamide): —C(═O)NR¹R²,wherein R¹ and R² are independently amino substituents, as defined foramino groups. Examples of amido groups include, but are not limited to,—C(═O)NH₂, —C(═O)NHCH₃, —C(═O)N(CH₃)₂, —C(═O)NHCH₂CH₃, and—C(═O)N(CH₂CH₃)₂, as well as amido groups in which R¹ and R², togetherwith the nitrogen atom to which they are attached, form a heterocyclicstructure as in, for example, piperidinocarbonyl, morpholinocarbonyl,thiomorpholinocarbonyl, and piperazinocarbonyl.

Thioamido (thiocarbamyl): —C(═S)NR¹R², wherein R¹ and R² areindependently amino substituents, as defined for amino groups. Examplesof amido groups include, but are not limited to, —C(═S)NH₂, —C(═S)NHCH₃,—C(═S)N(CH₃)₂, and —C(═S)NHCH₂CH₃.

Acylamido (acylamino): —NR¹C(═O)R², wherein R¹ is an amide substituent,for example, hydrogen, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group,or a C₅₋₂₀ aryl group, preferably hydrogen or a C₁₋₇ alkyl group, and R²is an acyl substituent, for example, a C₁₋₇ alkyl group, a C₃₋₂₀heterocyclyl group, or a C₅₋₂₀ aryl group, preferably hydrogen or a C₁₋₇alkyl group.

Examples of acylamide groups include, but are not limited to,—NHC(═O)CH₃, —NHC(═O)CH₂CH₃, and —NHC(═O)Ph. R¹ and R² may together forma cyclic structure, as in, for example, succinimidyl, maleimidyl, andphthalimidyl:

Aminocarbonyloxy: —OC(═O)NR¹R², wherein R¹ and R² are independentlyamino substituents, as defined for amino groups. Examples ofaminocarbonyloxy groups include, but are not limited to, —OC(═O)NH₂,—OC(═O)NHMe, —OC(═O)NMe₂, and —OC(═O)NEt₂.

Ureido: —N(R¹)CONR²R³ wherein R² and R³ are independently aminosubstituents, as defined for amino groups, and R¹ is a ureidosubstituent, for example, hydrogen, a C₁₋₇ alkyl group, a C₃₋₂₀heterocyclyl group, or a C₅₋₂₀ aryl group, preferably hydrogen or a C₁₋₇alkyl group. Examples of ureido groups include, but are not limited to,—NHCONH₂, —NHCONHMe, —NHCONHEt, —NHCONMe₂, —NHCONEt₂, —NMeCONH₂,—NMeCONHMe, —NMeCONHEt, —NMeCONMe₂, and —NMeCONEt₂.

Guanidino: —NH—C(═NH)NH₂.

Tetrazolyl: a five membered aromatic ring having four nitrogen atoms andone carbon atom,

Imino: ═NR, wherein R is an imino substituent, for example, for example,hydrogen, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀aryl group, preferably H or a C₁₋₇alkyl group. Examples of imino groupsinclude, but are not limited to, ═NH, ═NMe, and =NEt.

Amidine (amidino): —C(═NR)NR₂, wherein each R is an amidine substituent,for example, hydrogen, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group,or a C₅₋₂₀ aryl group, preferably H or a C₁₋₇ alkyl group. Examples ofamidine groups include, but are not limited to, —C(═NH)NH₂, —C(═NH)NMe₂,and —C(═NMe)NMe₂.

Nitro: —NO₂.

Nitroso: —NO.

Azido: —N₃.

Cyano (nitrile, carbonitrile): —CN.

Isocyano: —NC.

Cyanato: —OCN.

Isocyanato: —NCO.

Thiocyano (thiocyanato): —SCN.

Isothiocyano (isothiocyanato): —NCS.

Sulfhydryl (thiol, mercapto): —SH.

Thioether (sulfide): —SR, wherein R is a thioether substituent, forexample, a C₁₋₇ alkyl group (also referred to as a C₁₋₇alkylthio group),a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group, preferably a C₁₋₇alkyl group. Examples of C₁₋₇ alkylthio groups include, but are notlimited to, —SCH₃ and —SCH₂CH₃.

Disulfide: —SS—R, wherein R is a disulfide substituent, for example, aC₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group,preferably a C₁₋₇ alkyl group (also referred to herein as C₁₋₇ alkyldisulfide). Examples of C₁₋₇ alkyl disulfide groups include, but are notlimited to, —SSCH₃ and —SSCH₂CH₃.

Sulfine (sulfinyl, sulfoxide): —S(═O)R, wherein R is a sulfinesubstituent, for example, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclylgroup, or a C₅₋₂₀ aryl group, preferably a C₁₋₇ alkyl group. Examples ofsulfine groups include, but are not limited to, —S(═O)CH₃ and—S(═O)CH₂CH₃.

Sulfone (sulfonyl): —S(═O)₂R, wherein R is a sulfone substituent, forexample, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ arylgroup, preferably a C₁₋₇ alkyl group, including, for example, afluorinated or perfluorinated C₁₋₇ alkyl group. Examples of sulfonegroups include, but are not limited to, —S(═O)₂CH₃ (methanesulfonyl,mesyl), —S(═O)₂CF₃ (triflyl), —S(═O)₂CH₂CH₃ (esyl), —S(═O)₂C₄F₉(nonaflyl), —S(═O)₂CH₂CF₃ (tresyl), —S(═O)₂CH₂CH₂NH₂ (tauryl), —S(═O)₂Ph(phenylsulfonyl, besyl), 4-methylphenylsulfonyl (tosyl),4-chlorophenylsulfonyl (closyl), 4-bromophenylsulfonyl (brosyl),4-nitrophenyl (nosyl), 2-naphthalenesulfonate (napsyl), and5-dimethylamino-naphthalen-1-ylsulfonate (dansyl).

Sulfinic acid (sulfino): —S(═O)OH, —SO₂H.

Sulfonic acid (sulfo): —S(═O)₂OH, —SO₃H.

Sulfinate (sulfinic acid ester): —S(═O)OR; wherein R is a sulfinatesubstituent, for example, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclylgroup, or a C₅₋₂₀ aryl group, preferably a C₁₋₇ alkyl group. Examples ofsulfinate groups include, but are not limited to, —S(═O)OCH₃(methoxysulfinyl; methyl sulfinate) and —S(═O)OCH₂CH₃ (ethoxysulfinyl;ethyl sulfinate).

Sulfonate (sulfonic acid ester): —S(═O)₂OR, wherein R is a sulfonatesubstituent, for example, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclylgroup, or a C₅₋₂₀ aryl group, preferably a C₁₋₇ alkyl group. Examples ofsulfonate groups include, but are not limited to, —S(═O)₂OCH₃(methoxysulfonyl; methyl sulfonate) and —S(═O)₂OCH₂CH₃ (ethoxysulfonyl;ethyl sulfonate).

Sulfinyloxy: —OS(═O)R, wherein R is a sulfinyloxy substituent, forexample, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ arylgroup, preferably a C₁₋₇ alkyl group.

Examples of sulfinyloxy groups include, but are not limited to,—OS(═O)CH₃ and —OS(═O)CH₂CH₃.

Sulfonyloxy: —OS(═O)₂R, wherein R is a sulfonyloxy substituent, forexample, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ arylgroup, preferably a C₁₋₇ alkyl group. Examples of sulfonyloxy groupsinclude, but are not limited to, —OS(═O)₂CH₃ (mesylate) and—OS(═O)₂CH₂CH₃ (esylate).

Sulfate: —OS(═O)₂OR; wherein R is a sulfate substituent, for example, aC₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group,preferably a C₁₋₇ alkyl group. Examples of sulfate groups include, butare not limited to, —OS(═O)₂OCH₃ and —SO(═O)₂OCH₂CH₃.

Sulfamyl (sulfamoyl; sulfinic acid amide; sulfinamide): —S(═O)NR¹R²,wherein R¹ and R² are independently amino substituents, as defined foramino groups. Examples of sulfamyl groups include, but are not limitedto, —S(═O)NH₂, —S(═O)NH(CH₃), —S(═O)N(CH₃)₂, —S(═O)NH(CH₂CH₃),—S(═O)N(CH₂CH₃)₂, and —S(═O)NHPh.

Sulfonamido (sulfinamoyl; sulfonic acid amide; sulfonamide):—S(═O)₂NR¹R², wherein R¹ and R² are independently amino substituents, asdefined for amino groups. Examples of sulfonamido groups include, butare not limited to, —S(═O)₂NH₂, —S(═O)₂NH(CH₃), —S(═O)₂N(CH₃)₂,—S(═O)₂NH(CH₂CH₃), —S(═O)₂N(CH₂CH₃)₂, and —S(═O)₂NHPh.

Sulfamino: —NR¹S(═O)₂OH, wherein R¹ is an amino substituent, as definedfor amino groups. Examples of sulfamino groups include, but are notlimited to, —NHS(═O)₂OH and —N(CH₃)S(═O)₂OH.

Sulfonamino: —NR¹S(═O)₂R, wherein R¹ is an amino substituent, as definedfor amino groups, and R is a sulfonamino substituent, for example, aC₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group,preferably a C₁₋₇ alkyl group. Examples of sulfonamino groups include,but are not limited to, —NHS(═O)₂CH₃ and —N(CH₃)S(═O)₂C₆H₅.

Sulfinamino: —NR¹S(═O)R, wherein R¹ is an amino substituent, as definedfor amino groups, and R is a sulfinamino substituent, for example, aC₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group,preferably a C₁₋₇ alkyl group. Examples of sulfinamino groups include,but are not limited to, —NHS(═O)CH₃ and —N(CH₃)S(═O)C₆H₅.

Phosphino (phosphine): —PR₂, wherein R is a phosphino substituent, forexample, —H, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀aryl group, preferably —H, a C₁₋₇ alkyl group, or a C₅₋₂₀ aryl group.Examples of phosphino groups include, but are not limited to, —PH₂,—P(CH₃)₂, —P(CH₂CH₃)₂, —P(t-Bu)₂, and —P(Ph)₂.

Phospho: —P(═O)₂.

Phosphinyl (phosphine oxide): —P(═O)R₂, wherein R is a phosphinylsubstituent, for example, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclylgroup, or a C₅₋₂₀ aryl group, preferably a C₁₋₇ alkyl group or a C₅₋₂₀aryl group. Examples of phosphinyl groups include, but are not limitedto, —P(═O)(CH₃)₂, —P(═O)(CH₂CH₃)₂, —P(═O)(t-Bu)₂, and —P(═O)(Ph)₂.

Phosphonic acid (phosphono): —P(═O)(OH)₂.

Phosphonate (phosphono ester): —P(═O)(OR)₂, where R is a phosphonatesubstituent, for example, —H, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclylgroup, or a C₅₋₂₀ aryl group, preferably —H, a C₁₋₇ alkyl group, or aC₅₋₂₀ aryl group. Examples of phosphonate groups include, but are notlimited to, —P(═O)(OCH₃)₂, —P(═O)(OCH₂CH₃)₂, —P(═O)(O-t-Bu)₂, and—P(═O)(OPh)₂.

Phosphoric acid (phosphonooxy): —OP(═O)(OH)₂.

Phosphate (phosphonooxy ester): —OP(═O)(OR)₂, where R is a phosphatesubstituent, for example, —H, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclylgroup, or a C₅₋₂₀ aryl group, preferably —H, a C₁₋₇ alkyl group, or aC₅₋₂₀ aryl group. Examples of phosphate groups include, but are notlimited to, —OP(═O)(OCH₃)₂, —OP(═O)(OCH₂CH₃)₂, —OP(═O)(O-t-Bu)₂, and—OP(═O)(OPh)₂.

Phosphorous acid: —OP(OH)₂.

Phosphite: —OP(OR)₂, where R is a phosphite substituent, for example,—H, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ arylgroup, preferably —H, a C₁₋₇ alkyl group, or a C₅₋₂₀ aryl group.Examples of phosphite groups include, but are not limited to,—OP(OCH₃)₂, —OP(OCH₂CH₃)₂, —OP(O-t-Bu)₂, and —OP(OPh)₂.

Phosphoramidite: —OP(OR¹)—NR² ₂, where R¹ and R² are phosphoramiditesubstituents, for example, —H, a (optionally substituted) C₁₋₇ alkylgroup, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group, preferably —H,a C₁₋₇ alkyl group, or a C₅₋₂₀ aryl group. Examples of phosphoramiditegroups include, but are not limited to, —OP(OCH₂CH₃)—N(CH₃)₂,—OP(OCH₂CH₃)—N(i-Pr)₂, and —OP(OCH₂CH₂CN)—N(i-Pr)₂.

Phosphoramidate: —OP(═O)(OR¹)—NR² ₂, where R¹ and R² are phosphoramidatesubstituents, for example, —H, a (optionally substituted) C₁₋₇ alkylgroup, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group, preferably —H,a C₁₋₇ alkyl group, or a C₅₋₂₀ aryl group. Examples of phosphoramidategroups include, but are not limited to, —OP(═O)(OCH₂CH₃)—N(CH₃)₂,—OP(═O)(OCH₂CH₃)—N(i-Pr)₂, and —OP(═O)(OCH₂CH₂CN)—N(i-Pr)₂.

Alkylene

C₃₋₁₂ alkylene: The term “C₃₋₁₂ alkylene”, as used herein, pertains to abidentate moiety obtained by removing two hydrogen atoms, either bothfrom the same carbon atom, or one from each of two different carbonatoms, of a hydrocarbon compound having from 3 to 12 carbon atoms(unless otherwise specified), which may be aliphatic or alicyclic, andwhich may be saturated, partially unsaturated, or fully unsaturated.Thus, the term “alkylene” includes the sub-classes alkenylene,alkynylene, cycloalkylene, etc., discussed below.

Examples of linear saturated C₃₋₁₂ alkylene groups include, but are notlimited to, —(CH₂)_(n)— where n is an integer from 3 to 12, for example,—CH₂CH₂CH₂— (propylene), —CH₂CH₂CH₂CH₂— (butylene), —CH₂CH₂CH₂CH₂CH₂—(pentylene) and —CH₂CH₂CH₂CH-₂CH₂CH₂CH₂— (heptylene).

Examples of branched saturated C₃₋₁₂ alkylene groups include, but arenot limited to, —CH(CH₃)CH₂—, —CH(CH₃)CH₂CH₂—, —CH(CH₃)CH₂CH₂CH₂—,—CH₂CH(CH₃)CH₂—, —CH₂CH(CH₃)CH₂CH₂—, —CH(CH₂CH₃)—, —CH(CH₂CH₃)CH₂—, and—CH₂CH(CH₂CH₃)CH₂—.

Examples of linear partially unsaturated C₃₋₁₂ alkylene groups (C₃₋₁₂alkenylene, and alkynylene groups) include, but are not limited to,—CH═CH—CH₂—, —CH₂—CH═CH₂—, —CH═CH—CH₂—CH₂—, —CH═CH—CH₂—CH₂—CH₂—,—CH═CH—CH═CH—, —CH═CH—CH═CH—CH₂—, —CH═CH—CH═CH—CH₂—CH₂—,—CH═CH—CH₂—CH═CH—, —CH═CH—CH₂—CH₂—CH═CH—, and —CH₂—C≡C—CH₂—.

Examples of branched partially unsaturated C₃₋₁₂ alkylene groups (C₃₋₁₂alkenylene and alkynylene groups) include, but are not limited to,—C(CH₃)═CH—, —C(CH₃)═CH—CH₂—, —CH═CH—CH(CH₃)— and —C≡C—CH(CH₃)—.

Examples of alicyclic saturated C₃₋₁₂ alkylene groups (C₃₋₁₂cycloalkylenes) include, but are not limited to, cyclopentylene (e.g.cyclopent-1,3-ylene), and cyclohexylene (e.g. cyclohex-1,4-ylene).

Examples of alicyclic partially unsaturated C₃₋₁₂ alkylene groups (C₃₋₁₂cycloalkylenes) include, but are not limited to, cyclopentenylene (e.g.4-cyclopenten-1,3-ylene), cyclohexenylene (e.g. 2-cyclohexen-1,4-ylene;3-cyclohexen-1,2-ylene; 2,5-cyclohexadien-1,4-ylene).

Where the C₃₋₁₂ alkylene group is interrupted by a heteroatom, thesubscript refers to the number of atoms in the chain including theheteroatoms. For example, the chain —C₂H₄—O—C₂H₄— would be a C₅ group.

Where the C₃₋₁₂ alkylene group is interrupted by a heteroatom, thesubscript refers to the number of atoms directly in the chain includingthe aromatic ring. For example, the chain

would be a C₅ group.

Ligand Unit

The Ligand Unit may be of any kind, and include a protein, polypeptide,peptide and a non-peptidic agent that specifically binds to a targetmolecule. In some embodiments, the Ligand unit may be a protein,polypeptide or peptide. In some embodiments, the Ligand unit may be acyclic polypeptide. These Ligand units can include antibodies or afragment of an antibody that contains at least one targetmolecule-binding site, lymphokines, hormones, growth factors, or anyother cell binding molecule or substance that can specifically bind to atarget.

The terms “specifically binds” and “specific binding” refer to thebinding of an antibody or other protein, polypeptide or peptide to apredetermined molecule (e.g., an antigen). Typically, the antibody orother molecule binds with an affinity of at least about 1×10⁷ M⁻¹, andbinds to the predetermined molecule with an affinity that is at leasttwo-fold greater than its affinity for binding to a non-specificmolecule (e.g., BSA, casein) other than the predetermined molecule or aclosely-related molecule.

Examples of Ligand units include those agents described for use in WO2007/085930, which is incorporated herein.

In some embodiments, the Ligand unit is a Cell Binding Agent that bindsto an extracellular target on a cell. Such a Cell Binding Agent can be aprotein, polypeptide, peptide or a non-peptidic agent. In someembodiments, the Cell Binding Agent may be a protein, polypeptide orpeptide. In some embodiments, the Cell Binding Agent may be a cyclicpolypeptide. The Cell Binding Agent also may be antibody or anantigen-binding fragment of an antibody. Thus, in one embodiment, thepresent invention provides an antibody-drug conjugate (ADC).

Cell Binding Agent

A cell binding agent may be of any kind, and include peptides andnon-peptides. These can include antibodies or a fragment of an antibodythat contains at least one binding site, lymphokines, hormones, hormonemimetics, vitamins, growth factors, nutrient-transport molecules, or anyother cell binding molecule or substance.

Peptides

In one embodiment, the cell binding agent is a linear or cyclic peptidecomprising 4-30, preferably 6-20, contiguous amino acid residues. Inthis embodiment, it is preferred that one cell binding agent is linkedto one monomer or dimer pyrrolobenzodiazepine compound.

In one embodiment the cell binding agent comprises a peptide that bindsintegrin a_(v)36. The peptide may be selective for α_(v)β₆ over XYS.

In one embodiment the cell binding agent comprises the A20FMDV-Cyspolypeptide. The A20FMDV-Cys has the sequence: NAVPNLRGDLQVLAQKVARTC.Alternatively, a variant of the A20FMDV-Cys sequence may be used whereinone, two, three, four, five, six, seven, eight, nine or ten amino acidresidues are substituted with another amino acid residue. Furthermore,the polypeptide may have the sequence NAVXXXXXXXXXXXXXXXRTC.

Antibodies

The term “antibody” herein is used in the broadest sense andspecifically covers monoclonal antibodies, polyclonal antibodies,dimers, multimers, multispecific antibodies (e.g., bispecificantibodies), and antibody fragments, so long as they exhibit the desiredbiological activity (Miller et al (2003) Jour. of Immunology170:4854-4861). Antibodies may be murine, human, humanized, chimeric, orderived from other species. An antibody is a protein generated by theimmune system that is capable of recognizing and binding to a specificantigen. (Janeway, C., Travers, P., Walport, M., Shlomchik (2001) ImmunoBiology, 5th Ed., Garland Publishing, New York). A target antigengenerally has numerous binding sites, also called epitopes, recognizedby CDRs on multiple antibodies. Each antibody that specifically binds toa different epitope has a different structure. Thus, one antigen mayhave more than one corresponding antibody. An antibody includes afull-length immunoglobulin molecule or an immunologically active portionof a full-length immunoglobulin molecule, i.e., a molecule that containsan antigen binding site that immunospecifically binds an antigen of atarget of interest or part thereof, such targets including but notlimited to, cancer cell or cells that produce autoimmune antibodiesassociated with an autoimmune disease. The immunoglobulin can be of anytype (e.g. IgG, IgE, IgM, IgD, and IgA), class (e.g. IgG1, IgG2, IgG3,IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecule. Theimmunoglobulins can be derived from any species, including human,murine, or rabbit origin.

“Antibody fragments” comprise a portion of a full length antibody,generally the antigen binding or variable region thereof. Examples ofantibody fragments include Fab, Fab′, F(ab′)₂, and scFv fragments;diabodies; linear antibodies; fragments produced by a Fab expressionlibrary, anti-idiotypic (anti-Id) antibodies, CDR (complementarydetermining region), and epitope-binding fragments of any of the abovewhich immunospecifically bind to cancer cell antigens, viral antigens ormicrobial antigens, single-chain antibody molecules; and multispecificantibodies formed from antibody fragments.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies, i.e.the individual antibodies comprising the population are identical exceptfor possible naturally occurring mutations that may be present in minoramounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. Furthermore, in contrast to polyclonalantibody preparations which include different antibodies directedagainst different determinants (epitopes), each monoclonal antibody isdirected against a single determinant on the antigen. In addition totheir specificity, the monoclonal antibodies are advantageous in thatthey may be synthesized uncontaminated by other antibodies. The modifier“monoclonal” indicates the character of the antibody as being obtainedfrom a substantially homogeneous population of antibodies, and is not tobe construed as requiring production of the antibody by any particularmethod. For example, the monoclonal antibodies to be used in accordancewith the present invention may be made by the hybridoma method firstdescribed by Kohler et al (1975) Nature 256:495, or may be made byrecombinant DNA methods (see, U.S. Pat. No. 4,816,567). The monoclonalantibodies may also be isolated from phage antibody libraries using thetechniques described in Clackson et al (1991) Nature, 352:624-628; Markset al (1991) J. Mol. Biol., 222:581-597 or from transgenic mice carryinga fully human immunoglobulin system (Lonberg (2008) Curr. Opinion20(4):450-459).

The monoclonal antibodies herein specifically include “chimeric”antibodies in which a portion of the heavy and/or light chain isidentical with or homologous to corresponding sequences in antibodiesderived from a particular species or belonging to a particular antibodyclass or subclass, while the remainder of the chain(s) is identical withor homologous to corresponding sequences in antibodies derived fromanother species or belonging to another antibody class or subclass, aswell as fragments of such antibodies, so long as they exhibit thedesired biological activity (U.S. Pat. No. 4,816,567; and Morrison et al(1984) Proc. Natl. Acad. Sci. USA, 81:6851-6855). Chimeric antibodiesinclude “primatized” antibodies comprising variable domainantigen-binding sequences derived from a non-human primate (e.g. OldWorld Monkey or Ape) and human constant region sequences.

An “intact antibody” herein is one comprising a VL and VH domains, aswell as a light chain constant domain (CL) and heavy chain constantdomains, CH1, CH2 and CH3. The constant domains may be native sequenceconstant domains (e.g. human native sequence constant domains) or aminoacid sequence variant thereof. The intact antibody may have one or more“effector functions” which refer to those biological activitiesattributable to the Fc region (a native sequence Fc region or amino acidsequence variant Fc region) of an antibody. Examples of antibodyeffector functions include Clq binding; complement dependentcytotoxicity; Fc receptor binding; antibody-dependent cell-mediatedcytotoxicity (ADCC); phagocytosis; and down regulation of cell surfacereceptors such as B cell receptor and BCR.

Depending on the amino acid sequence of the constant domain of theirheavy chains, intact antibodies can be assigned to different “classes.”There are five major classes of intact antibodies: IgA, IgD, IgE, IgG,and IgM, and several of these may be further divided into “subclasses”(isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy-chainconstant domains that correspond to the different classes of antibodiesare called a, b, E, y, and p, respectively. The subunit structures andthree-dimensional configurations of different classes of immunoglobulinsare well known.

Humanisation

Techniques to reduce the in vivo immunogenicity of a non-human antibodyor antibody fragment include those termed “humanisation”.

A “humanized antibody” refers to a polypeptide comprising at least aportion of a modified variable region of a human antibody wherein aportion of the variable region, preferably a portion substantially lessthan the intact human variable domain, has been substituted by thecorresponding sequence from a non-human species and wherein the modifiedvariable region is linked to at least another part of another protein,preferably the constant region of a human antibody. The expression“humanized antibodies” includes human antibodies in which one or morecomplementarity determining region (“CDR”) amino acid residues and/orone or more framework region (“FW” or “FR”) amino acid residues aresubstituted by amino acid residues from analogous sites in rodent orother non-human antibodies. The expression “humanized antibody” alsoincludes an immunoglobulin amino acid sequence variant or fragmentthereof that comprises an FR having substantially the amino acidsequence of a human immunoglobulin and a CDR having substantially theamino acid sequence of a non-human immunoglobulin.

“Humanized” forms of non-human (e.g., murine) antibodies are chimericantibodies that contain minimal sequence derived from non-humanimmunoglobulin. Or, looked at another way, a humanized antibody is ahuman antibody that also contains selected sequences from non-human(e.g. murine) antibodies in place of the human sequences. A humanizedantibody can include conservative amino acid substitutions ornon-natural residues from the same or different species that do notsignificantly alter its binding and/or biologic activity. Suchantibodies are chimeric antibodies that contain minimal sequence derivedfrom non-human immunoglobulins.

There are a range of humanisation techniques, including ‘CDR grafting’,‘guided selection’, ‘deimmunization’, ‘resurfacing’ (also known as‘veneering’), ‘composite antibodies’, ‘Human String ContentOptimisation’ and framework shuffling.

CDR Grafting

In this technique, the humanized antibodies are human immunoglobulins(recipient antibody) in which residues from a complementary-determiningregion (CDR) of the recipient antibody are replaced by residues from aCDR of a non-human species (donor antibody) such as mouse, rat, camel,bovine, goat, or rabbit having the desired properties (in effect, thenon-human CDRs are ‘grafted’ onto the human framework). In someinstances, framework region (FR) residues of the human immunoglobulinare replaced by corresponding non-human residues (this may happen when,for example, a particular FR residue has significant effect on antigenbinding).

Furthermore, humanized antibodies can comprise residues that are foundneither in the recipient antibody nor in the imported CDR or frameworksequences. These modifications are made to further refine and maximizeantibody performance. Thus, in general, a humanized antibody willcomprise all of at least one, and in one aspect two, variable domains,in which all or all of the hypervariable loops correspond to those of anon-human immunoglobulin and all or substantially all of the FR regionsare those of a human immunoglobulin sequence. The humanized antibodyoptionally also will comprise at least a portion of an immunoglobulinconstant region (Fc), or that of a human immunoglobulin.

Guided Selection

The method consists of combining the V_(H) or V_(L) domain of a givennon-human antibody specific for a particular epitope with a human V_(H)or V_(L) library and specific human V domains are selected against theantigen of interest. This selected human VH is then combined with a VLlibrary to generate a completely human VH×VL combination. The method isdescribed in Nature Biotechnology (N.Y.) 12, (1994) 899-903.

Composite Antibodies

In this method, two or more segments of amino acid sequence from a humanantibody are combined within the final antibody molecule. They areconstructed by combining multiple human VH and VL sequence segments incombinations which limit or avoid human T cell epitopes in the finalcomposite antibody V regions. Where required, T cell epitopes arelimited or avoided by, exchanging V region segments contributing to orencoding a T cell epitope with alternative segments which avoid T cellepitopes. This method is described in US 2008/0206239 A1.

Deimmunization

This method involves the removal of human (or other second species)T-cell epitopes from the V regions of the therapeutic antibody (or othermolecule). The therapeutic antibodies V-region sequence is analysed forthe presence of MHC class II-binding motifs by, for example, comparisonwith databases of MHC-binding motifs (such as the “motifs” databasehosted at www.wehi.edu.au). Alternatively, MHC class II-binding motifsmay be identified using computational threading methods such as thosedevised by Altuvia et al. (J. Mol. Biol. 249 244-250 (1995)); in thesemethods, consecutive overlapping peptides from the V-region sequencesare testing for their binding energies to MHC class II proteins. Thisdata can then be combined with information on other sequence featureswhich relate to successfully presented peptides, such as amphipathicity,Rothbard motifs, and cleavage sites for cathepsin B and other processingenzymes.

Once potential second species (e.g. human) T-cell epitopes have beenidentified, they are eliminated by the alteration of one or more aminoacids. The modified amino acids are usually within the T-cell epitopeitself, but may also be adjacent to the epitope in terms of the primaryor secondary structure of the protein (and therefore, may not beadjacent in the primary structure). Most typically, the alteration is byway of substitution but, in some circumstances amino acid addition ordeletion will be more appropriate.

All alterations can be accomplished by recombinant DNA technology, sothat the final molecule may be prepared by expression from a recombinanthost using well established methods such as Site Directed Mutagenesis.However, the use of protein chemistry or any other means of molecularalteration is also possible.

Resurfacing

This method involves:

-   -   (a) determining the conformational structure of the variable        region of the non-human (e.g. rodent) antibody (or fragment        thereof) by constructing a three-dimensional model of the        non-human antibody variable region;    -   (b) generating sequence alignments using relative accessibility        distributions from x-ray crystallographic structures of a        sufficient number of non-human and human antibody variable        region heavy and light chains to give a set of heavy and light        chain framework positions wherein the alignment positions are        identical in 98% of the sufficient number of non-human antibody        heavy and light chains;    -   (c) defining for the non-human antibody to be humanized, a set        of heavy and light chain surface exposed amino acid residues        using the set of framework positions generated in step (b);    -   (d) identifying from human antibody amino acid sequences a set        of heavy and light chain surface exposed amino acid residues        that is most closely identical to the set of surface exposed        amino acid residues defined in step (c), wherein the heavy and        light chain from the human antibody are or are not naturally        paired;    -   (e) substituting, in the amino acid sequence of the non-human        antibody to be humanized, the set of heavy and light chain        surface exposed amino acid residues defined in step (c) with the        set of heavy and light chain surface exposed amino acid residues        identified in step (d);    -   (f) constructing a three-dimensional model of the variable        region of the non-human antibody resulting from the substituting        specified in step (e);    -   (g) identifying, by comparing the three-dimensional models        constructed in steps (a) and (f), any amino acid residues from        the sets identified in steps (c) or (d), that are within 5        Angstroms of any atom of any residue of the complementarity        determining regions of the non-human antibody to be humanized;        and    -   (h) changing any residues identified in step (g) from the human        to the original non-human amino acid residue to thereby define a        non-human antibody humanizing set of surface exposed amino acid        residues; with the proviso that step (a) need not be conducted        first, but must be conducted prior to step (g).

Superhumanization

The method compares the non-human sequence with the functional humangermline gene repertoire. Those human genes encoding canonicalstructures identical or closely related to the non-human sequences areselected. Those selected human genes with highest homology within theCDRs are chosen as FR donors. Finally, the non-human CDRs are graftedonto these human FRs. This method is described in patent WO 2005/079479A2.

Human String Content Optimization

This method compares the non-human (e.g. mouse) sequence with therepertoire of human germline genes and the differences are scored asHuman String Content (HSC) that quantifies a sequence at the level ofpotential MHC/T-cell epitopes. The target sequence is then humanized bymaximizing its HSC rather than using a global identity measure togenerate multiple diverse humanized variants (described in MolecularImmunology, 44, (2007) 1986-1998).

Framework Shuffling

The CDRs of the non-human antibody are fused in-frame to cDNA poolsencompassing all known heavy and light chain human germline geneframeworks. Humanised antibodies are then selected by e.g. panning ofthe phage displayed antibody library. This is described in Methods 36,43-60 (2005).

Examples of cell binding agents include those agents described for usein WO 2007/085930, which is incorporated herein.

Tumour-associate antigens and cognate antibodies for use in embodimentsof the present invention are listed below.

Tumor-Associated Antigens and Cognate Antibodies (1) BMPRIB (BoneMorphogenetic Protein Receptor-Type IB) Nucleotide

Genbank accession no. NM_001203

Genbank version no. NM_001203.2 GI: 169790809

Genbank record update date: Sep. 23, 2012 02:06 PM

Polypeptide

Genbank accession no. NP_001194

Genbank version no. NP_001194.1 GI: 4502431

Genbank record update date: Sep. 23, 2012 02:06 PM

Cross-References

ten Dijke, P., et al Science 264 (5155): 101-104 (1994), Oncogene 14(11):1377-1382 (1997)); WO2004/063362 (Claim 2); WO2003/042661 (Claim12); US2003/134790-A1 (Page 38-39); WO2002/102235 (Claim 13; Page 296);WO2003/055443 (Page 91-92); WO2002/99122 (Example 2; Page 528-530);WO2003/029421 (Claim 6); WO2003/024392 (Claim 2; FIG. 112); WO2002/98358(Claim 1; Page 183); WO2002/54940 (Page 100-101); WO2002/59377 (Page349-350); WO2002/30268 (Claim 27; Page 376); WO2001/48204 (Example; FIG.4); NP_001194 bone morphogenetic protein receptor, typeIB/pid=NP_001194.1; MIM: 603248; AY065994

(2) E16 (LAT1, SLC7A5) Nucleotide

Genbank accession no. NM_003486

Genbank version no. NM_003486.5 GI: 71979931

Genbank record update date: Jun. 27, 2012 12:06 PM

Polypeptide

Genbank accession no. NP_003477

Genbank version no. NP_003477.4 GI: 71979932

Genbank record update date: Jun. 27, 2012 12:06 PM

Cross References

Biochem. Biophys. Res. Commun. 255 (2), 283-288 (1999), Nature 395(6699):288-291 (1998), Gaugitsch, H. W., et al (1992) J. Biol. Chem. 267(16):11267-11273); WO2004/048938 (Example 2); WO2004/032842 (ExampleIV); WO2003/042661 (Claim 12); WO2003/016475 (Claim 1); WO2002/78524(Example 2); WO2002/99074 (Claim 19; Page 127-129); WO2002/86443 (Claim27; Pages 222, 393); WO2003/003906 (Claim 10; Page 293); WO2002/64798(Claim 33; Page 93-95); WO2000/14228 (Claim 5; Page 133-136);US2003/224454 (FIG. 3); WO2003/025138 (Claim 12; Page 150); NP_003477solute carrier family 7 (cationic amino acid transporter, y+system),member 5/pid=NP_003477.3—Homo sapiens; MIM: 600182; NM_015923.

(3) STEAP1 (Six Transmembrane Epithelial Antigen of Prostate) Nucleotide

Genbank accession no. NM_012449

Genbank version no. NM_012449.2 GI: 22027487

Genbank record update date: Sep. 9, 2012 02:57 PM

Polypeptide

Genbank accession no. NP_036581

Genbank version no. NP_036581.1 GI: 9558759

Genbank record update date: Sep. 9, 2012 02:57 PM

Cross References

Cancer Res. 61 (15), 5857-5860 (2001), Hubert, R. S., et al (1999) Proc.Natl. Acad. Sci. U.S.A. 96 (25):14523-14528); WO2004/065577 (Claim 6);WO2004/027049 (FIG. 1L); EP1394274 (Example 11); WO2004/016225 (Claim2); WO2003/042661 (Claim 12); US2003/157089 (Example 5); US2003/185830(Example 5); US2003/064397 (FIG. 2); WO2002/89747 (Example 5; Page618-619); WO2003/022995 (Example 9; FIG. 13A, Example 53; Page 173,Example 2; FIG. 2A); six transmembrane epithelial antigen of theprostate; MIM: 604415.

(4) 0772P (CA125, MUC16) Nucleotide

Genbank accession no. AF361486

Genbank version no. AF361486.3 GI: 34501466

Genbank record update date: Mar. 11, 2010 07:56 AM

Polypeptide

Genbank accession no. AAK74120

Genbank version no. AAK74120.3 GI: 34501467

Genbank record update date: Mar. 11, 2010 07:56 AM

Cross References

J. Biol. Chem. 276 (29):27371-27375 (2001)); WO2004/045553 (Claim 14);WO2002/92836 (Claim 6; FIG. 12); WO2002/83866 (Claim 15; Page 116-121);US2003/124140 (Example 16); GI: 34501467;

(5) MPF (MPF, MSLN, SMR, Megakaryocyte Potentiating Factor, Mesothelin)Nucleotide

Genbank accession no. NM_005823

Genbank version no. NM_005823.5 GI: 293651528

Genbank record update date: Sep. 2, 2012 01:47 PM

Polypeptide

Genbank accession no. NP_005814

Genbank version no. NP_005814.2 GI: 53988378

Genbank record update date: Sep. 2, 2012 01:47 PM

Cross References

Yamaguchi, N., et al Biol. Chem. 269 (2), 805-808 (1994), Proc. Natl.Acad. Sci. U.S.A. 96 (20):11531-11536 (1999), Proc. Natl. Acad. Sci.U.S.A. 93 (1):136-140 (1996), J. Biol. Chem. 270 (37):21984-21990(1995)); WO2003/101283 (Claim 14); (WO2002/102235 (Claim 13; Page287-288); WO2002/101075 (Claim 4; Page 308-309); WO2002/71928 (Page320-321); WO94/10312 (Page 52-57); IM: 601051.

(6) Napi3b (NAPI-3B, NPTIIb, SLC34A2, Solute Carrier Family 34 (SodiumPhosphate), Member 2, Type II Sodium-Dependent Phosphate Transporter 3b)Nucleotide

Genbank accession no. NM_006424

Genbank version no. NM_006424.2 GI: 110611905

Genbank record update date: Jul. 22, 2012 03:39 PM

Polypeptide

Genbank accession no. NP_006415

Genbank version no. NP_006415.2 GI: 110611906

Genbank record update date: Jul. 22, 2012 03:39 PM

Cross References

J. Biol. Chem. 277 (22):19665-19672 (2002), Genomics 62 (2):281-284(1999), Feild, J. A., et al (1999) Biochem. Biophys. Res. Commun. 258(3):578-582); WO2004/022778 (Claim 2); EP1394274 (Example 11);WO2002/102235 (Claim 13; Page 326); EP0875569 (Claim 1; Page 17-19);WO2001/57188 (Claim 20; Page 329); WO2004/032842 (Example IV);WO2001/75177 (Claim 24; Page 139-140); MIM: 604217.

(7) Sema 5b (FLJ10372, KIAA1445, Mm.42015, SEMA5B, SEMAG, Semaphorin 5bHlog, Sema Domain, Seven Thrombospondin Repeats (Type 1 and Type1-Like), Transmembrane Domain™ and Short Cytoplasmic Domain,(Semaphorin) 5B) Nucleotide

Genbank accession no. AB040878

Genbank version no. AB040878.1 GI: 7959148

Genbank record update date: Aug. 2, 2006 05:40 PM

Polypeptide

Genbank accession no. BAA95969

Genbank version no. BAA95969.1 GI: 7959149

Genbank record update date: Aug. 2, 2006 05:40 PM

Cross References

Nagase T., et al (2000) DNA Res. 7 (2):143-150); WO2004/000997 (Claim1); WO2003/003984 (Claim 1); WO2002/06339 (Claim 1; Page 50);WO2001/88133 (Claim 1; Page 41-43, 48-58); WO2003/054152 (Claim 20);WO2003/101400 (Claim 11); Accession: Q9P283; Genew; HGNC: 10737

(8) PSCA hIg (2700050C12Rik, C530008016Rik, RIKEN cDNA 2700050C12, RIKENcDNA 2700050C12 gene)

Nucleotide

Genbank accession no. AY358628

Genbank version no. AY358628.1 GI: 37182377

Genbank record update date: Dec. 1, 2009 04:15 AM

Polypeptide

Genbank accession no. AAQ88991

Genbank version no. AAQ88991.1 GI: 37182378

Genbank record update date: Dec. 1, 2009 04:15 AM

Cross References

Ross et al (2002) Cancer Res. 62:2546-2553; US2003/129192 (Claim 2);US2004/044180 (Claim 12); US2004/044179 (Claim 11); US2003/096961 (Claim11); US2003/232056 (Example 5); WO2003/105758 16 (Claim 12);US2003/206918 (Example 5); EP1347046 (Claim 1); WO2003/025148 (Claim20); GI: 37182378.

(9) ETBR (Endothelin Type B Receptor) Nucleotide

Genbank accession no. AY275463

Genbank version no. AY275463.1 GI: 30526094

Genbank record update date: Mar. 11, 2010 02:26 AM

Polypeptide

Genbank accession no. AAP32295

Genbank version no. AAP32295.1 GI: 30526095

Genbank record update date: Mar. 11, 2010 02:26 AM

Cross References

Nakamuta M., et al Biochem. Biophys. Res. Commun. 177, 34-39, 1991;Ogawa Y., et al Biochem. Biophys. Res. Commun. 178, 248-255, 1991; AraiH., et al Jpn. Circ. J. 56, 1303-1307, 1992; Arai H., et al J. Biol.Chem. 268, 3463-3470, 1993; Sakamoto A., Yanagisawa M., et al Biochem.Biophys. Res. Commun. 178, 656-663, 1991; Elshourbagy N. A., et al J.Biol. Chem. 268, 3873-3879, 1993; Haendler B., et al J. Cardiovasc.Pharmacol. 20, s1-S4, 1992; Tsutsumi M., et al Gene 228, 43-49, 1999;Strausberg R. L., et al Proc. Natl. Acad. Sci. U.S.A. 99, 16899-16903,2002; Bourgeois C., et al J. Clin. Endocrinol. Metab. 82, 3116-3123,1997; Okamoto Y., et al Biol. Chem. 272, 21589-21596, 1997; Verheij J.B., et al Am. J. Med. Genet. 108, 223-225, 2002; Hofstra R. M. W., et alEur. J. Hum. Genet. 5, 180-185, 1997; Puffenberger E. G., et al Cell 79,1257-1266, 1994; Attie T., et al, Hum. Mol. Genet. 4, 2407-2409, 1995;Auricchio A., et al Hum. Mol. Genet. 5:351-354, 1996; Amiel J., et alHum. Mol. Genet. 5, 355-357, 1996; Hofstra R. M. W., et al Nat. Genet.12, 445-447, 1996; Svensson P. J., et al Hum. Genet. 103, 145-148, 1998;Fuchs S., et al Mol. Med. 7, 115-124, 2001; Pingault V., et al (2002)Hum. Genet. 111, 198-206; WO2004/045516 (Claim 1); WO2004/048938(Example 2); WO2004/040000 (Claim 151); WO2003/087768 (Claim 1);WO2003/016475 (Claim 1); WO2003/016475 (Claim 1); WO2002/61087 (FIG. 1);WO2003/016494 (FIG. 6); WO2003/025138 (Claim 12; Page 144); WO2001/98351(Claim 1; Page 124-125); EP0522868 (Claim 8; FIG. 2); WO2001/77172(Claim 1; Page 297-299); US2003/109676; U.S. Pat. No. 6,518,404 (FIG.3); U.S. Pat. No. 5,773,223 (Claim 1a; Col 31-34); WO2004/001004.

(10) MSG783 (RNF124, Hypothetical Protein FLJ20315) Nucleotide

Genbank accession no. NM_017763

Genbank version no. NM_017763.4 GI: 167830482

Genbank record update date: Jul. 22, 2012 12:34 AM

Polypeptide

Genbank accession no. NP_060233

Genbank version no. NP_060233.3 GI: 56711322

Genbank record update date: Jul. 22, 2012 12:34 AM

Cross References

WO2003/104275 (Claim 1); WO2004/046342 (Example 2); WO2003/042661 (Claim12); WO2003/083074 (Claim 14; Page 61); WO2003/018621 (Claim 1);WO2003/024392 (Claim 2; FIG. 93); WO2001/66689 (Example 6); LocusID:54894.

(11) STEAP2 (HGNC_8639, IPCA-1, PCANAP1, STAMP1, STEAP2, STMP, ProstateCancer Associated Gene 1, Prostate Cancer Associated Protein 1, SixTransmembrane Epithelial Antigen of Prostate 2, Six TransmembraneProstate Protein) Nucleotide

Genbank accession no. AF455138

Genbank version no. AF455138.1 GI: 22655487

Genbank record update date: Mar. 11, 2010 01:54 AM

Polypeptide

Genbank accession no. AAN04080

Genbank version no. AAN04080.1 GI: 22655488

Genbank record update date: Mar. 11, 2010 01:54 AM

Cross References

Lab. Invest. 82 (11):1573-1582 (2002)); WO2003/087306; US2003/064397(Claim 1; FIG. 1); WO2002/72596 (Claim 13; Page 54-55); WO2001/72962(Claim 1; FIG. 4B); WO2003/104270 (Claim 11); WO2003/104270 (Claim 16);US2004/005598 (Claim 22); WO2003/042661 (Claim 12); US2003/060612 (Claim12; FIG. 10); WO2002/26822 (Claim 23; FIG. 2); WO2002/16429 (Claim 12;FIG. 10); GI: 22655488.

(12) TrpM4 (BR22450, FLJ20041, TRPM4, TRPM4B, Transient ReceptorPotential Cation Channel, Subfamily M, Member 4) Nucleotide

Genbank accession no. NM_017636

Genbank version no. NM_017636.3 GI: 304766649

Genbank record update date: Jun. 29, 2012 11:27 AM

Polypeptide

Genbank accession no. NP_060106

Genbank version no. NP_060106.2 GI: 21314671

Genbank record update date: Jun. 29, 2012 11:27 AM

Cross References

Xu, X. Z., et al Proc. Natl. Acad. Sci. U.S.A. 98 (19):10692-10697(2001), Cell 109 (3):397-407 (2002), J. Biol. Chem. 278 (33):30813-30820(2003)); US2003/143557 (Claim 4); WO2000/40614 (Claim 14; Page 100-103);WO2002/10382 (Claim 1; FIG. 9A); WO2003/042661 (Claim 12); WO2002/30268(Claim 27; Page 391); US2003/219806 (Claim 4); WO2001/62794 (Claim 14;FIG. 1A-D); MIM: 606936.

(13) CRIPTO (CR, CR1, CRGF, CRIPTO, TDGFI, Teratocarcinoma-DerivedGrowth Factor) Nucleotide

Genbank accession no. NM_003212

Genbank version no. NM_003212.3 GI: 292494881

Genbank record update date: Sep. 23, 2012 02:27 PM

Polypeptide

Genbank accession no. NP_003203

Genbank version no. NP_003203.1 GI: 4507425

Genbank record update date: Sep. 23, 2012 02:27 PM

Cross References

Ciccodicola, A., et al EMBO J. 8 (7):1987-1991 (1989), Am. J. Hum.Genet. 49 (3):555-565 (1991)); US2003/224411 (Claim 1); WO2003/083041(Example 1); WO2003/034984 (Claim 12); WO2002/88170 (Claim 2; Page52-53); WO2003/024392 (Claim 2; FIG. 58); WO2002/16413 (Claim 1; Page94-95, 105); WO2002/22808 (Claim 2; FIG. 1); U.S. Pat. No. 5,854,399(Example 2; Col 17-18); U.S. Pat. No. 5,792,616 (FIG. 2); MIM: 187395.

(14) CD21 (CR2 (Complement Receptor 2) or C3DR (C3d/Epstein Barr VirusReceptor) or Hs.73792) Nucleotide

Genbank accession no M26004

Genbank version no. M26004.1 GI: 181939

Genbank record update date: Jun. 23, 2010 08:47 AM

Polypeptide

Genbank accession no. AAA35786

Genbank version no. AAA35786.1 GI: 181940

Genbank record update date: Jun. 23, 2010 08:47 AM

Cross References

Fujisaku et al (1989) J. Biol. Chem. 264 (4):2118-2125); Weis J. J., etal J. Exp. Med. 167, 1047-1066, 1988; Moore M., et al Proc. Natl. Acad.Sci. U.S.A. 84, 9194-9198, 1987; Barel M., et al Mol. Immunol. 35,1025-1031, 1998; Weis J. J., et al Proc. Natl. Acad. Sci. U.S.A. 83,5639-5643, 1986; Sinha S. K., et al (1993) J. Immunol. 150, 5311-5320;WO2004/045520 (Example 4); US2004/005538 (Example 1); WO2003/062401(Claim 9); WO2004/045520 (Example 4); WO91/02536 (FIGS. 9.1-9.9);WO2004/020595 (Claim 1); Accession: P20023; Q13866; Q14212; EMBL;M26004; AAA35786.1.

(15) CD79b (CD79B, CD793, IGb (immunoglobulin-associated beta), B29)

Nucleotide

Genbank accession no NM_000626

Genbank version no. NM_000626.2 GI: 90193589

Genbank record update date: Jun. 26, 2012 01:53 PM

Polypeptide

Genbank accession no. NP_000617

Genbank version no. NP_000617.1 GI: 11038674

Genbank record update date: Jun. 26, 2012 01:53 PM

Cross References

Proc. Natl. Acad. Sci. U.S.A. (2003) 100 (7):4126-4131, Blood (2002) 100(9):3068-3076, Muller et al (1992) Eur. J. Immunol. 22 (6):1621-1625);WO2004/016225 (claim 2, FIG. 140); WO2003/087768, US2004/101874 (claim1, page 102); WO2003/062401 (claim 9); WO2002/78524 (Example 2);US2002/150573 (claim 5, page 15); U.S. Pat. No. 5,644,033; WO2003/048202(claim 1, pages 306 and 309); WO 99/58658, U.S. Pat. No. 6,534,482(claim 13, FIG. 17A/B); WO2000/55351 (claim 11, pages 1145-1146); MIM:147245

(16) FcRH2 (IFGP4, IRTA4, SPAP1A (SH2 Domain Containing PhosphataseAnchor Protein 1a), SPAP1B, SPAP1C) Nucleotide

Genbank accession no NM_030764

Genbank version no. NM_030764.3 GI: 227430280

Genbank record update date: Jun. 30, 2012 12:30 AM

Polypeptide

Genbank accession no. NP_110391

Genbank version no. NP_110391.2 GI: 19923629

Genbank record update date: Jun. 30, 2012 12:30 AM

Cross References

AY358130); Genome Res. 13 (10):2265-2270 (2003), Immunogenetics 54(2):87-95 (2002), Blood 99 (8):2662-2669 (2002), Proc. Natl. Acad. Sci.U.S.A. 98 (17):9772-9777 (2001), Xu, M. J., et al (2001) Biochem.Biophys. Res. Commun. 280 (3):768-775; WO2004/016225 (Claim 2);WO2003/077836; WO2001/38490 (Claim 5; FIG. 18D-1-18D-2); WO2003/097803(Claim 12); WO2003/089624 (Claim 25); MIM: 606509.

(17) HER2 (ErbB2) Nucleotide

Genbank accession no M11730

Genbank version no. M11730.1 GI: 183986

Genbank record update date: Jun. 23, 2010 08:47 AM

Polypeptide

Genbank accession no. AAA75493

Genbank version no. AAA75493.1 GI: 306840

Genbank record update date: Jun. 23, 2010 08:47 AM

Cross References

Coussens L., et al Science (1985) 230(4730):1132-1139); Yamamoto T., etal Nature 319, 230-234, 1986; Semba K., et al Proc. Natl. Acad. Sci.U.S.A. 82, 6497-6501, 1985; Swiercz J. M., et al J. Cell Biol. 165,869-880, 2004; Kuhns J. J., et al J. Biol. Chem. 274, 36422-36427, 1999;Cho H.-S., et al Nature 421, 756-760, 2003; Ehsani A., et al (1993)Genomics 15, 426-429; WO2004/048938 (Example 2); WO2004/027049 (FIG.11); WO2004/009622; WO2003/081210; WO2003/089904 (Claim 9);WO2003/016475 (Claim 1); US2003/118592; WO2003/008537 (Claim 1);WO2003/055439 (Claim 29; FIG. 1A-B); WO2003/025228 (Claim 37; FIG. 5C);WO2002/22636 (Example 13; Page 95-107); WO2002/12341 (Claim 68; FIG. 7);WO2002/13847 (Page 71-74); WO2002/14503 (Page 114-117); WO2001/53463(Claim 2; Page 41-46); WO2001/41787 (Page 15); WO2000/44899 (Claim 52;FIG. 7); WO2000/20579 (Claim 3; FIG. 2); U.S. Pat. No. 5,869,445 (Claim3; Col 31-38); WO9630514 (Claim 2; Page 56-61); EP1439393 (Claim 7);WO2004/043361 (Claim 7); WO2004/022709; WO2001/00244 (Example 3; FIG.4); Accession: P04626; EMBL; M11767; AAA35808.1. EMBL; M11761;AAA35808.1

Antibodies Abbott: US20110177095

-   -   For example, an antibody comprising CDRs having overall at least        80% sequence identity to CDRs having amino acid sequences of SEQ        ID NO:3 (CDR-H1), SEQ ID NO:4 (CDR-H2), SEQ ID NO:5 (CDR-H3),        SEQ ID NO:104 and/or SEQ ID NO:6 (CDR-L1), SEQ ID NO:7 (CDR-L2),        and SEQ ID NO:8 (CDR-L3), wherein the anti-HER2 antibody or        anti-HER2 binding fragment has reduced immunogenicity as        compared to an antibody having a VH of SEQ ID NO:1 and a VL of        SEQ ID NO:2.

Biogen: US20100119511

-   -   For example, ATCC accession numbers: PTA-10355, PTA-10356,        PTA-10357, PTA-10358    -   For example, a purified antibody molecule that binds to HER2        comprising a all six CDR's from an antibody selected from the        group consisting of BIIB71F10 (SEQ ID NOs:11, 13), BIIB69A09        (SEQ ID NOs:15, 17); BIIB67F10 (SEQ ID NOs:19, 21); BIIB67F11        (SEQ ID NOs:23, 25), BIIB66A12 (SEQ ID NOs:27, 29), BIIB66C01        (SEQ ID NOs:31, 33), BIIB65C10 (SEQ ID NOs:35, 37), BIIB65H09        (SEQ ID NOs:39, 41) and BIIB65B03 (SEQ ID NOs:43, 45), or CDRs        which are identical or which have no more than two alterations        from said CDRs.

Herceptin (Genentech)—U.S. Pat. No. 6,054,297; ATCC accession no.CRL-10463 (Genentech)

Pertuzumab (Genentech)

-   -   US20110117097        -   for example, see SEQ IDs No. 15&16, SEQ IDs No. 17&18, SEQ            IDs No. 23&24 & ATCC accession numbers HB-12215, HB-12216,            CRL 10463, HB-12697.    -   US20090285837    -   US20090202546        -   for example, ATCC accession numbers: HB-12215, HB-12216, CRL            10463, HB-12698.    -   US20060088523        -   for example, ATCC accession numbers: HB-12215, HB-12216        -   for example, an antibody comprising the variable light and            variable heavy amino acid sequences in SEQ ID Nos. 3 and 4,            respectively.        -   for example, an antibody comprising a light chain amino acid            sequence selected from SEQ ID No. 15 and 23, and a heavy            chain amino acid sequence selected from SEQ ID No. 16 and 24    -   US20060018899        -   for example, ATCC accession numbers: (7C2) HB-12215, (7F3)            HB-12216, (4D5) CRL-10463, (2C4) HB-12697.        -   for example, an antibody comprising the amino acid sequence            in SEQ ID No. 23, or a deamidated and/or oxidized variant            thereof.    -   US2011/0159014        -   for example, an antibody having a light chain variable            domain comprising the hypervariable regions of SEQ ID NO:            1”.        -   For example, an antibody having a heavy chain variable            domain comprising the hypervariable regions of SEQ ID NO: 2.    -   US20090187007        Glycotope: TrasGEX antibody http://www.glycotope.com/pipeline    -   For example, see International Joint Cancer Institute and        Changhai Hospital Cancer Cent: HMTI-Fc Ab—Gao J., et al BMB Rep.        2009 Oct. 31; 42(10):636-41.

Symphogen: US20110217305 Union Stem Cell & Gene Engineering, China—Liu HQ., et al Xi Bao Yu Fen Zi Mian YiXue Za Zhi. 2010 May; 26(5):456-8.(18) NCA (CEACAM6) Nucleotide

Genbank accession no M18728

Genbank version no. M18728.1 GI: 189084

Genbank record update date: Jun. 23, 2010 08:48 AM

Polypeptide

Genbank accession no. AAA59907

Genbank version no. AAA59907.1 GI: 189085

Genbank record update date: Jun. 23, 2010 08:48 AM

Cross References

Barnett T., et al Genomics 3, 59-66, 1988; Tawaragi Y., et al Biochem.Biophys. Res. Commun. 150, 89-96, 1988; Strausberg R. L., et al Proc.Natl. Acad. Sci. U.S.A. 99:16899-16903, 2002; WO2004/063709; EP1439393(Claim 7); WO2004/044178 (Example 4); WO2004/031238; WO2003/042661(Claim 12); WO2002/78524 (Example 2); WO2002/86443 (Claim 27; Page 427);WO2002/60317 (Claim 2); Accession: P40199; Q14920; EMBL; M29541;AAA59915.1. EMBL; M18728.

(19) MDP (DPEP1) Nucleotide

Genbank accession no BC017023

Genbank version no. BC017023.1 GI: 16877538

Genbank record update date: Mar. 6, 2012 01:00 PM

Polypeptide

Genbank accession no. AAH17023

Genbank version no. AAH17023.1 GI: 16877539

Genbank record update date: Mar. 6, 2012 01:00 PM

Cross References

Proc. Natl. Acad. Sci. U.S.A. 99 (26):16899-16903 (2002)); WO2003/016475(Claim 1); WO2002/64798 (Claim 33; Page 85-87); JP05003790 (FIG. 6-8);WO99/46284 (FIG. 9); MIM: 179780.

(20) IL20R-Alpha (IL20Ra, ZCYTOR7) Nucleotide

Genbank accession no AF184971

Genbank version no. AF184971.1 GI: 6013324

Genbank record update date: Mar. 10, 2010 10:00 PM

Polypeptide

Genbank accession no. AAF01320

Genbank version no. AAF01320.1 GI: 6013325

Genbank record update date: Mar. 10, 2010 10:00 PM

Cross References

Clark H. F., et al Genome Res. 13, 2265-2270, 2003; Mungall A. J., et alNature 425, 805-811, 2003; Blumberg H., et al Cell 104, 9-19, 2001;Dumoutier L., et al J. Immunol. 167, 3545-3549, 2001; Parrish-Novak J.,et al J. Biol. Chem. 277, 47517-47523, 2002; Pletnev S., et al (2003)Biochemistry 42:12617-12624; Sheikh F., et al (2004) J. Immunol. 172,2006-2010; EP1394274 (Example 11); US2004/005320 (Example 5);WO2003/029262 (Page 74-75); WO2003/002717 (Claim 2; Page 63);WO2002/22153 (Page 45-47); US2002/042366 (Page 20-21); WO2001/46261(Page 57-59); WO2001/46232 (Page 63-65); WO98/37193 (Claim 1; Page55-59); Accession: Q9UHF4; Q6UWA9; Q96SH8; EMBL; AF184971; AAF01320.1.

(21) Brevican (BCAN, BEHAB) Nucleotide

Genbank accession no AF229053

Genbank version no. AF229053.1 GI: 10798902

Genbank record update date: Mar. 11, 2010 12:58 AM

Polypeptide

Genbank accession no. AAG23135

Genbank version no. AAG23135.1 GI: 10798903

Genbank record update date: Mar. 11, 2010 12:58 AM

Cross References

Gary S. C., et al Gene 256, 139-147, 2000; Clark H. F., et al GenomeRes. 13, 2265-2270, 2003; Strausberg R. L., et al Proc. Natl. Acad. Sci.U.S.A. 99, 16899-16903, 2002; US2003/186372 (Claim 11); US2003/186373(Claim 11); US2003/119131 (Claim 1; FIG. 52); US2003/119122 (Claim 1;FIG. 52); US2003/119126 (Claim 1); US2003/119121 (Claim 1; FIG. 52);US2003/119129 (Claim 1); US2003/119130 (Claim 1); US2003/119128 (Claim1; FIG. 52); US2003/119125 (Claim 1); WO2003/016475 (Claim 1);WO2002/02634 (Claim 1)

(22) EphB2R (DRT, ERK, Hek5, EPHT3, Tyro5) Nucleotide

Genbank accession no NM_004442

Genbank version no. NM_004442.6 GI: 111118979

Genbank record update date: Sep. 8, 2012 04:43 PM

Polypeptide

Genbank accession no. NP_004433

Genbank version no. NP_004433.2 GI: 21396504

Genbank record update date: Sep. 8, 2012 04:43 PM

Cross References

Chan, J. and Watt, V. M., Oncogene 6 (6), 1057-1061 (1991) Oncogene 10(5):897-905 (1995), Annu. Rev. Neurosci. 21:309-345 (1998), Int. Rev.Cytol. 196:177-244 (2000)); WO2003042661 (Claim 12); WO200053216 (Claim1; Page 41); WO2004065576 (Claim 1); WO2004020583 (Claim 9);WO2003004529 (Page 128-132); WO200053216 (Claim 1; Page 42); MIM:600997.

(23) ASLG659 (B7h) Nucleotide

Genbank accession no. AX092328

Genbank version no. AX092328.1 GI: 13444478

Genbank record update date: Jan. 26, 2011 07:37 AM

Cross References

US2004/0101899 (Claim 2); WO2003104399 (Claim 11); WO2004000221 (FIG.3); US2003/165504 (Claim 1); US2003/124140 (Example 2); US2003/065143(FIG. 60); WO2002/102235 (Claim 13; Page 299); US2003/091580 (Example2); WO2002/10187 (Claim 6; FIG. 10); WO2001/94641 (Claim 12; FIG. 7b);WO2002/02624 (Claim 13; FIG. 1A-1B); US2002/034749 (Claim 54; Page45-46); WO2002/06317 (Example 2; Page 320-321, Claim 34; Page 321-322);WO2002/71928 (Page 468-469); WO2002/02587 (Example 1; FIG. 1);WO2001/40269 (Example 3; Pages 190-192); WO2000/36107 (Example 2; Page205-207); WO2004/053079 (Claim 12); WO2003/004989 (Claim 1);WO2002/71928 (Page 233-234, 452-453); WO 01/16318.

(24) PSCA (Prostate Stem Cell Antigen Precursor) Nucleotide

Genbank accession no AJ297436

Genbank version no. AJ297436.1 GI: 9367211

Genbank record update date: Feb. 1, 2011 11:25 AM

Polypeptide

Genbank accession no. CAB97347

Genbank version no. CAB97347.1 GI: 9367212

Genbank record update date: Feb. 1, 2011 11:25 AM

Cross References

Reiter R. E., et al Proc. Natl. Acad. Sci. U.S.A. 95, 1735-1740, 1998;Gu Z., et al Oncogene 19, 1288-1296, 2000; Biochem. Biophys. Res.Commun. (2000) 275(3):783-788; WO2004/022709; EP1394274 (Example 11);US2004/018553 (Claim 17); WO2003/008537 (Claim 1); WO2002/81646 (Claim1; Page 164); WO2003/003906 (Claim 10; Page 288); WO2001/40309 (Example1; FIG. 17); US2001/055751 (Example 1; FIG. 1b); WO2000/32752 (Claim 18;FIG. 1); WO98/51805 (Claim 17; Page 97); WO98/51824 (Claim 10; Page 94);WO98/40403 (Claim 2; FIG. 1B); Accession: 043653; EMBL; AF043498;AAC39607.1

(25) GEDA Nucleotide

Genbank accession no AY260763

Genbank version no. AY260763.1 GI: 30102448

Genbank record update date: Mar. 11, 2010 02:24 AM

Polypeptide

Genbank accession no. AAP14954

Genbank version no. AAP14954.1 GI: 30102449

Genbank record update date: Mar. 11, 2010 02:24 AM

Cross References

AP14954 lipoma HMGIC fusion-partnerlike protein/pid=AAP14954.1—Homosapiens (human); WO2003/054152 (Claim 20); WO2003/000842 (Claim 1);WO2003/023013 (Example 3, Claim 20); US2003/194704 (Claim 45); GI:30102449;

(26) BAFF-R (B Cell-Activating Factor Receptor, BLyS Receptor 3, BR3)Nucleotide

Genbank accession no AF116456

Genbank version no. AF116456.1 GI: 4585274

Genbank record update date: Mar. 10, 2010 09:44 PM

Polypeptide

Genbank accession no. AAD25356

Genbank version no. AAD25356.1 GI: 4585275

Genbank record update date: Mar. 10, 2010 09:44 PM

Cross References

BAFF receptor/pid=NP_443177.1—Homo sapiens: Thompson, J. S., et alScience 293 (5537), 2108-2111 (2001); WO2004/058309; WO2004/011611;WO2003/045422 (Example; Page 32-33); WO2003/014294 (Claim 35; FIG. 6B);WO2003/035846 (Claim 70; Page 615-616); WO2002/94852 (Col 136-137);WO2002/38766 (Claim 3; Page 133); WO2002/24909 (Example 3; FIG. 3); MIM:606269; NP_443177.1; NM_052945_1; AF132600

(27) CD22 (B-Cell Receptor CD22-B Isoform, BL-CAM, Lyb-8, Lyb8,SIGLEC-2, FLJ22814) Nucleotide

Genbank accession no AK026467

Genbank version no. AK026467.1 GI: 10439337

Genbank record update date: Sep. 11, 2006 11:24 PM

Polypeptide

Genbank accession no. BAB15489

Genbank version no. BAB15489.1 GI: 10439338

Genbank record update date: Sep. 11, 2006 11:24 PM

Cross References

Wilson et al (1991) J. Exp. Med. 173:137-146; WO2003/072036 (Claim 1;FIG. 1); IM: 107266; NP_001762.1; NM_001771_1.

(27a) Cd22 (Cd22 Molecule) Nucleotide

Genbank accession no X52785

Genbank version no. X52785.1 GI: 29778

Genbank record update date: Feb. 2, 2011 10:09 AM

Polypeptide

Genbank accession no. CAA36988

Genbank version no. CAA36988.1 GI: 29779

Genbank record update date: Feb. 2, 2011 10:09 AM

Cross References

Stamenkovic I. et al., Nature 345 (6270), 74-77 (1990)

Other Information

Official Symbol: CD22

Other Aliases: SIGLEC-2, SIGLEC2

Other Designations: B-cell receptor CD22; B-lymphocyte cell adhesionmolecule; BL-CAM; CD22 antigen; T-cell surface antigen Leu-14; sialicacid binding Ig-like lectin 2; sialic acid-binding Ig-like lectin 2

Antibodies

G5/44 (Inotuzumab): DiJoseph J F., et al Cancer Immunol Immunother. 2005January; 54(1):11-24.

Epratuzumab—Goldenberg D M., et al Expert Rev Anticancer Ther. 6(10):1341-53, 2006.

(28) CD79a (CD79A, CD79alpha), Immunoglobulin-Associated Alpha, a BCell-Specific Protein that Covalently Interacts with Ig Beta (CD79B) andForms a Complex on the Surface with Ig M Molecules, Transduces a SignalInvolved in B-Cell Differentiation), pI: 4.84, MW: 25028 TM: 2 [P] GeneChromosome: 19q13.2).

Nucleotide

Genbank accession no NM_001783

Genbank version no. NM_001783.3 GI: 90193587

Genbank record update date: Jun. 26, 2012 01:48 PM

Polypeptide

Genbank accession no. NP_001774

Genbank version no. NP_001774.1 GI: 4502685

Genbank record update date: Jun. 26, 2012 01:48 PM

Cross References

WO2003/088808, US2003/0228319; WO2003/062401 (claim 9); US2002/150573(claim 4, pages 13-14); WO99/58658 (claim 13, FIG. 16); WO92/07574 (FIG.1); U.S. Pat. No. 5,644,033; Ha et al (1992) J. Immunol.148(5):1526-1531; Müller et al (1992) Eur. J. Immunol. 22:1621-1625;Hashimoto et al (1994) Immunogenetics 40(4):287-295; Preud'homme et al(1992) Clin. Exp. Immunol. 90(1):141-146; Yu et al (1992) J. Immunol.148(2) 633-637; Sakaguchi et al (1988) EMBO J. 7(11):3457-3464

(29) CXCR5 (Burkitt's Lymphoma Receptor 1, a G Protein-Coupled Receptorthat is Activated by the CXCL13 Chemokine, Functions in LymphocyteMigration and Humoral Defense, Plays a Role in HIV-2 Infection andPerhaps Development of AIDS, Lymphoma, Myeloma, and Leukemia); 372 aa,pI: 8.54 MW: 41959 TM: 7[P] Gene Chromosome: 11q23.3,

Nucleotide

Genbank accession no NM_001716

Genbank version no. NM_001716.4 GI: 342307092

Genbank record update date: Sep. 30, 2012 01:49 PM

Polypeptide

Genbank accession no. NP_001707

Genbank version no. NP_001707.1 GI: 4502415

Genbank record update date: Sep. 30, 2012 01:49 PM

Cross References

WO2004/040000; WO2004/015426; US2003/105292 (Example 2); U.S. Pat. No.6,555,339 (Example 2); WO2002/61087 (FIG. 1); WO2001/57188 (Claim 20,page 269); WO2001/72830 (pages 12-13); WO2000/22129 (Example 1, pages152-153, Example 2, pages 254-256); WO99/28468 (claim 1, page 38); U.S.Pat. No. 5,440,021 (Example 2, col 49-52); WO94/28931 (pages 56-58);WO92/17497 (claim 7, FIG. 5); Dobner et al (1992) Eur. J. Immunol.22:2795-2799; Barella et al (1995) Biochem. J. 309:773-779

(30) HLA-DOB (Beta Subunit of MHC Class II Molecule (Ia Antigen) thatBinds Peptides and Presents them to CD4+T Lymphocytes); 273 aa, pI:6.56, MW: 30820. TM: 1 [P] Gene Chromosome: 6p21.3)

Nucleotide

Genbank accession no NM_002120

Genbank version no. NM_002120.3 GI: 118402587

Genbank record update date: Sep. 8, 2012 04:46 PM

Polypeptide

Genbank accession no. NP_002111

Genbank version no. NP_002111.1 GI: 4504403

Genbank record update date: Sep. 8, 2012 04:46 PM

Cross References

Tonnelle et al (1985) EMBO J. 4(11):2839-2847; Jonsson et al (1989)Immunogenetics 29(6):411-413; Beck et al (1992) J. Mol. Biol.228:433-441; Strausberg et al (2002) Proc. Natl. Acad. Sci USA99:16899-16903; Servenius et al (1987) J. Biol. Chem. 262:8759-8766;Beck et al (1996) J. Mol. Biol. 255:1-13; Naruse et al (2002) TissueAntigens 59:512-519; WO99/58658 (claim 13, FIG. 15); U.S. Pat. No.6,153,408 (Col 35-38); U.S. Pat. No. 5,976,551 (col 168-170); U.S. Pat.No. 6,011,146 (col 145-146); Kasahara et al (1989) Immunogenetics30(1):66-68; Larhammar et al (1985) J. Biol. Chem. 260(26):14111-14119

(31) P2X5 (Purinergic Receptor P2X Ligand-Gated Ion Channel 5, an IonChannel Gated by Extracellular ATP, May be Involved in SynapticTransmission and Neurogenesis, Deficiency May Contribute to thePathophysiology of Idiopathic Detrusor Instability); 422 aa), pI: 7.63,MW: 47206 TM: 1 [P] Gene Chromosome: 17p13.3).

Nucleotide

Genbank accession no NM_002561

Genbank version no. NM_002561.3 GI: 325197202

Genbank record update date: Jun. 27, 2012 12:41 AM

Polypeptide

Genbank accession no. NP_002552

Genbank version no. NP_002552.2 GI: 28416933

Genbank record update date: Jun. 27, 2012 12:41 AM

Cross References

Le et al (1997) FEBS Lett. 418(1-2):195-199; WO2004/047749;WO2003/072035 (claim 10); Touchman et al (2000) Genome Res. 10:165-173;WO2002/22660 (claim 20); WO2003/093444 (claim 1); WO2003/087768 (claim1); WO2003/029277 (page 82)

(32) CD72 (B-Cell Differentiation Antigen CD72, Lyb-2); 359 aa, pI:8.66, MW: 40225, TM: 1 [P] Gene Chromosome: 9p13.3). Nucleotide

Genbank accession no NM_001782

Genbank version no. NM_001782.2 GI: 194018444

Genbank record update date: Jun. 26, 2012 01:43 PM

Polypeptide

Genbank accession no. NP_001773

Genbank version no. NP_001773.1 GI: 4502683

Genbank record update date: Jun. 26, 2012 01:43 PM

Cross References

WO2004042346 (claim 65); WO2003/026493 (pages 51-52, 57-58);WO2000/75655 (pages 105-106); Von Hoegen et al (1990) J. Immunol.144(12):4870-4877; Strausberg et al (2002) Proc. Natl. Acad. Sci USA99:16899-16903.

(33) LY64 (Lymphocyte Antigen 64 (RP105), Type I Membrane Protein of theLeucine Rich Repeat (LRR) Family, Regulates B-Cell Activation andApoptosis, Loss of Function is Associated with Increased DiseaseActivity in Patients with Systemic Lupus Erythematosis); 661 aa, pI:6.20, MW: 74147 TM: 1 [P] Gene Chromosome: 5q12).

Nucleotide

Genbank accession no NM_005582

Genbank version no. NM_005582.2 GI: 167555126

Genbank record update date: Sep. 2, 2012 01:50 PM

Polypeptide

Genbank accession no. NP_005573

Genbank version no. NP_005573.2 GI: 167555127

Genbank record update date: Sep. 2, 2012 01:50 PM

Cross References

US2002/193567; WO97/07198 (claim 11, pages 39-42); Miura et al (1996)Genomics 38(3):299-304; Miura et al (1998) Blood 92:2815-2822;WO2003/083047; WO97/44452 (claim 8, pages 57-61); WO2000/12130 (pages24-26).

(34) FcRH1 (Fc Receptor-Like Protein 1, a Putative Receptor for theImmunoglobulin Fc Domain that Contains C2 Type Ig-Like and ITAM Domains,May have a Role in B-Lymphocyte Differentiation); 429 aa, pI: 5.28, MW:46925 TM: 1 [P] Gene Chromosome: 1q21-1q22)

Nucleotide

Genbank accession no NM_052938

Genbank version no. NM_052938.4 GI: 226958543

Genbank record update date: Sep. 2, 2012 01:43 PM

Polypeptide

Genbank accession no. NP_443170

Genbank version no. NP_443170.1 GI: 16418419

Genbank record update date: Sep. 2, 2012 01:43 PM

Cross References

WO2003/077836; WO2001/38490 (claim 6, FIG. 18E-1-18-E-2); Davis et al(2001) Proc. Natl. Acad. Sci USA 98(17):9772-9777; WO2003/089624 (claim8); EP1347046 (claim 1); WO2003/089624 (claim 7).

(35) IRTA2 (Immunoglobulin Superfamily Receptor Translocation Associated2, a Putative Immunoreceptor with Possible Roles in B Cell Developmentand Lymphomagenesis; Deregulation of the Gene by Translocation Occurs inSome B Cell Malignancies); 977 aa, pI: 6.88, MW: 106468, TM: 1 [P] GeneChromosome: 1q21)

Nucleotide

Genbank accession no AF343662

Genbank version no. AF343662.1 GI: 13591709

Genbank record update date: Mar. 11, 2010 01:16 AM

Polypeptide

Genbank accession no. AAK31325

Genbank version no. AAK31325.1 GI: 13591710

Genbank record update date: Mar. 11, 2010 01:16 AM

Cross References

AF343663, AF343664, AF343665, AF369794, AF397453, AK090423, AK090475,AL834187, AY358085; Mouse: AK089756, AY158090, AY506558; NP_112571.1;WO2003/024392 (claim 2, FIG. 97); Nakayama et al (2000) Biochem.Biophys. Res. Commun. 277(1):124-127; WO2003/077836; WO2001/38490 (claim3, FIG. 18B-1-18B-2).

(36) TENB2 (TMEFF2, Tomoregulin, TPEF, HPPI, TR, Putative TransmembraneProteoglycan, Related to the EGF/Heregulin Family of Growth Factors andFollistatin); 374 aa) Nucleotide

Genbank accession no AF179274

Genbank version no. AF179274.2 GI: 12280939

Genbank record update date: Mar. 11, 2010 01:05 AM

Polypeptide

Genbank accession no. AAD55776

Genbank version no. AAD55776.2 GI: 12280940

Genbank record update date: Mar. 11, 2010 01:05 AM

Cross References

NCBI Accession: AAD55776, AAF91397, AAG49451, NCBI RefSeq: NP_057276;NCBI Gene: 23671; OMIM: 605734; SwissProt Q9UIK5; AY358907, CAF85723,CQ782436; WO2004/074320; JP2004113151; WO2003/042661; WO2003/009814;EP1295944 (pages 69-70); WO2002/30268 (page 329); WO2001/90304;US2004/249130; US2004/022727; WO2004/063355; US2004/197325;US2003/232350; US2004/005563; US2003/124579; Horie et al (2000) Genomics67:146-152; Uchida et al (1999) Biochem. Biophys. Res. Commun.266:593-602; Liang et al (2000) Cancer Res. 60:4907-12; Glynne-Jones etal (2001) Int J Cancer. October 15; 94(2):178-84.

(37) PSMA-FOLH1 (Folate hydrolase (prostate-specific membrane antigen)1)

Nucleotide

Genbank accession no M99487

Genbank version no. M99487.1 GI: 190663

Genbank record update date: Jun. 23, 2010 08:48 AM

Polypeptide

Genbank accession no. AAA60209

Genbank version no. AAA60209.1 GI: 190664

Genbank record update date: Jun. 23, 2010 08:48 AM

Cross References

Israeli R. S., et al Cancer Res. 53 (2), 227-230 (1993)

Other Information

Official Symbol: FOLH1

Other Aliases: GIG27, FGCP, FOLH, GCP2, GCPII, NAALAD1, NAALAdase, PSM,PSMA, mGCP

Other Designations: N-acetylated alpha-linked acidic dipeptidase 1;N-acetylated-alpha-linked acidic dipeptidase I; NAALADase I; cellgrowth-inhibiting gene 27 protein; folylpoly-gamma-glutamatecarboxypeptidase; glutamate carboxylase II; glutamate carboxypeptidase2; glutamate carboxypeptidase II; membrane glutamate carboxypeptidase;prostate specific membrane antigen variant F;pteroylpoly-gamma-glutamate carboxypeptidase

Antibodies

U.S. Pat. No. 7,666,425: Antibodies produces by Hybridomas having thefollowing ATCC references: ATCC accession No. HB-12101, ATCC accessionNo. HB-12109, ATCC accession No. HB-12127 and ATCC accession No.HB-12126.

Proscan: a monoclonal antibody selected from the group consisting of8H12, 3E11, 17G1, 29B4, 30C1 and 20F2 (U.S. Pat. No. 7,811,564; MoffettS., et al Hybridoma (Larchmt). 2007 December; 26(6):363-72).

Cytogen: monoclonal antibodies 7E11-C5 (ATCC accession No. HB 10494) and9H10-A4 (ATCC accession No. HB11430)—U.S. Pat. No. 5,763,202

GlycoMimetics: NUH2—ATCC accession No. HB 9762 (U.S. Pat. No. 7,135,301)

Human Genome Science: HPRAJ70—ATCC accession No. 97131 (U.S. Pat. No.6,824,993); Amino acid sequence encoded by the cDNA clone (HPRAJ70)deposited as American Type Culture Collection (“ATCC”) Deposit No. 97131

Medarex: Anti-PSMA antibodies that lack fucosyl residues—U.S. Pat. No.7,875,278

Mouse anti-PSMA antibodies include the 3F5.4G6, 3D7.1.1, 4E10-1.14,3E11, 4D8, 3E6, 3C9, 2C7, 1G3, 3C4, 3C6, 4D4, 1G9, 5C8B9, 3G6, 4C8B9,and monoclonal antibodies. Hybridomas secreting 3F5.4G6, 3D7.1.1,4E10-1.14, 3E11, 4D8, 3E6, 3C9, 2C7, 1G3, 3C4, 3C6, 4D4, 1G9, 5C8B9, 3G6or 4C8B9 have been publicly deposited and are described in U.S. Pat. No.6,159,508. Relevant hybridomas have been publicly deposited and aredescribed in U.S. Pat. No. 6,107,090. Moreover, humanized anti-PSMAantibodies, including a humanized version of J591, are described infurther detail in PCT Publication WO 02/098897.

Other mouse anti-human PSMA antibodies have been described in the art,such as mAb 107-1A4 (Wang, S. et al. (2001) Int. J. Cancer 92:871-876)and mAb 2C9 (Kato, K. et al. (2003) Int. J. Urol. 10:439-444).

Examples of human anti-PSMA monoclonal antibodies include the 4A3, 7F12,8C12, 8A11, 16F9, 2A10, 2C6, 2F5 and 1C3 antibodies, isolated andstructurally characterized as originally described in PCT PublicationsWO 01/09192 and WO 03/064606 and in U.S. Provisional Application Ser.No. 60/654,125, entitled “Human Monoclonal Antibodies to ProstateSpecific Membrane Antigen (PSMA)”, filed on Feb. 18, 2005. The V.sub.Hamino acid sequences of 4A3, 7F12, 8C12, 8A11, 16F9, 2A10, 2C6, 2F5 and1C3 are shown in SEQ ID NOs: 1-9, respectively. The V.sub.L amino acidsequences of 4A3, 7F12, 8C12, 8A11, 16F9, 2A10, 2C6, 2F5 and 1C3 areshown in SEQ ID NOs: 10-18, respectively.

Other human anti-PSMA antibodies include the antibodies disclosed in PCTPublication WO 03/034903 and US Application No. 2004/0033229.

NW Biotherapeutics: A hybridoma cell line selected from the groupconsisting of 3F5.4G6 having ATCC accession number HB12060, 3D7-1.1.having ATCC accession number HB12309, 4E10-1.14 having ATCC accessionnumber HB12310, 3E11 (ATCC HB12488), 4D8 (ATCC HB12487), 3E6 (ATCCHB12486), 3C9 (ATCC HB12484), 2C7 (ATCC HB12490), 1G3 (ATCC HB12489),3C4 (ATCC HB12494), 3C6 (ATCC HB12491), 4D4 (ATCC HB12493), 1G9 (ATCCHB12495), 5C8B9 (ATCC HB12492) and 3G6 (ATCC HB12485)—see U.S. Pat. No.6,150,508

PSMA Development Company/Progenics/Cytogen—Seattle Genetics: mAb 3.9,produced by the hybridoma deposited under ATCC Accession No. PTA-3258 ormAb 10.3, produced by the hybridoma deposited under ATCC Accession No.PTA-3347—U.S. Pat. No. 7,850,971

PSMA Development Company—Compositions of PSMA antibodies (US20080286284, Table 1)

-   -   This application is a divisional of U.S. patent application Ser.        No. 10/395,894, filed on Mar. 21, 2003 (U.S. Pat. No. 7,850,971)

University Hospital Freiburg, Germany—mAbs 3/A12, 3/E7, and 3/F11 (WolfP., et al Prostate. 2010 Apr. 1; 70(5):562-9).

(38) SST (Somatostatin Receptor; Note that there are 5 Subtypes)

(38.1) SSTR2 (Somatostatin Receptor 2) Nucleotide

Genbank accession no NM_001050

Genbank version no. NM_001050.2 GI: 44890054

Genbank record update date: Aug. 19, 2012 01:37 PM

Polypeptide

Genbank accession no. NP_001041

Genbank version no. NP_001041.1 GI: 4557859

Genbank record update date: Aug. 19, 2012 01:37 PM

Cross References

Yamada Y., et al Proc. Natl. Acad. Sci. U.S.A. 89 (1), 251-255 (1992);Susini C., et al Ann Oncol. 2006 December; 17(12):1733-42

Other Information

Official Symbol: SSTR2

Other Designations: SRIF-1; SS2R; somatostatin receptor type 2

(38.2) SSTR5 (Somatostatin Receptor 5) Nucleotide

Genbank accession no D16827

Genbank version no. D16827.1 GI: 487683

Genbank record update date: Aug. 1, 2006 12:45 PM

Polypeptide

Genbank accession no. BAA04107

Genbank version no. BAA04107.1 GI: 487684

Genbank record update date: Aug. 1, 2006 12:45 PM

Cross References

Yamada, Y., et al Biochem. Biophys. Res. Commun. 195 (2), 844-852 (1993)

Other Information

Official Symbol: SSTR5

Other Aliases: SS-5-R

Other Designations: Somatostatin receptor subtype 5; somatostatinreceptor type 5

(38.3) SSTR1 (38.4) SSTR3 (38.5) SSTR4 AvB6—Both Subunits (39+40)

(39) ITGAV (Integrin, alpha V;

Nucleotide

Genbank accession no M14648 J02826 M18365

Genbank version no. M14648.1 GI: 340306

Genbank record update date: Jun. 23, 2010 08:56 AM

Polypeptide

Genbank accession no. AAA36808

Genbank version no. AAA36808.1 GI: 340307

Genbank record update date: Jun. 23, 2010 08:56 AM

Cross References

Suzuki S., et al Proc. Natl. Acad. Sci. U.S.A. 83 (22), 8614-8618 (1986)

Other Information

Official Symbol: ITGAV

Other Aliases: CD51, MSK8, VNRA, VTNR

Other Designations: antigen identified by monoclonal antibody L230;integrin alpha-V; integrin alphaVbeta3; integrin, alpha V (vitronectinreceptor, alpha polypeptide, antigen CD51); vitronectin receptor subunitalpha

(40) ITGB6 (Integrin, Beta 6) Nucleotide

Genbank accession no NM_000888

Genbank version no. NM_000888.3 GI: 9966771

Genbank record update date: Jun. 27, 2012 12:46 AM

Polypeptide

Genbank accession no. NP_000879

Genbank version no. NP_000879.2 GI: 9625002

Genbank record update date: Jun. 27, 2012 12:46 AM

Cross References

Sheppard D. J., et al Biol. Chem. 265 (20), 11502-11507 (1990)

Other Information

Official Symbol: ITGB6

Other Designations: integrin beta-6

Antibodies

Biogen: U.S. Pat. No. 7,943,742—Hybridoma clones 6.3G9 and 6.8G6 weredeposited with the ATCC, accession numbers ATCC PTA-3649 and -3645,respectively.

Biogen: U.S. Pat. No. 7,465,449—In some embodiments, the antibodycomprises the same heavy and light chain polypeptide sequences as anantibody produced by hybridoma 6.1A8, 6.3G9, 6.8G6, 6.2B1, 6.2B10,6.2A1, 6.2E5, 7.1G10, 7.7G5, or 7.1C5.

Centocor (J&J): U.S. Pat. Nos. 7,550,142; 7,163,681

-   -   For example in U.S. Pat. No. 7,550,142—an antibody having human        heavy chain and human light chain variable regions comprising        the amino acid sequences shown in SEQ ID NO: 7 and SEQ ID NO: 8.

Seattle Genetics: 15H3 (Ryan M C., et al Cancer Res Apr. 15, 2012; 72(8Supplement): 4630)

(41) CEACAM5 (Carcinoembryonic Antigen-Related Cell Adhesion Molecule 5)Nucleotide

Genbank accession no M17303

Genbank version no. M17303.1 GI: 178676

Genbank record update date: Jun. 23, 2010 08:47 AM

Polypeptide

Genbank accession no. AAB59513

Genbank version no. AAB59513.1 GI: 178677

Genbank record update date: Jun. 23, 2010 08:47 AM

Cross References

Beauchemin N., et al Mol. Cell. Biol. 7 (9), 3221-3230 (1987)

Other Information

Official Symbol: CEACAM5

Other Aliases: CD66e, CEA

Other Designations: meconium antigen 100

Antibodies

AstraZeneca-MedImmune: US 20100330103; US20080057063;

-   -   US20020142359        -   for example an antibody having complementarity determining            regions (CDRs) with the following sequences: heavy chain;            CDR1—DNYMH, CDR2—WIDPENGDTE YAPKFRG, CDR3—LIYAGYLAMD Y; and            light chain CDR1—SASSSVTYMH, CDR2—STSNLAS, CDR3—QQRSTYPLT.        -   Hybridoma 806.077 deposited as European Collection of Cell            Cultures (ECACC) deposit no. 96022936.

Research Corporation Technologies, Inc.: U.S. Pat. No. 5,047,507

Bayer Corporation: U.S. Pat. No. 6,013,772

BioAlliance: U.S. Pat. Nos. 7,982,017; 7,674,605

-   -   U.S. Pat. No. 7,674,605        -   an antibody comprising the heavy chain variable region            sequence from the amino acid sequence of SEQ ID NO: 1, and            the light chain variable region sequence from the amino acid            sequence of SEQ ID NO:2.        -   an antibody comprising the heavy chain variable region            sequence from the amino acid sequence of SEQ ID NO:5, and            the light chain variable region sequence from the amino acid            sequence of SEQ ID NO:6.

Celltech Therapeutics Limited: U.S. Pat. No. 5,877,293

The Dow Chemical Company: U.S. Pat. Nos. 5,472,693; 6,417,337; 6,333,405

-   -   U.S. Pat. No. 5,472,693—for example, ATCC No. CRL-11215    -   U.S. Pat. No. 6,417,337—for example, ATCC CRL-12208    -   U.S. Pat. No. 6,333,405—for example, ATCC CRL-12208

Immunomedics, Inc: U.S. Pat. Nos. 7,534,431; 7,230,084; 7,300,644;6,730,300;

-   -   US20110189085        -   an antibody having CDRs of the light chain variable region            comprise: CDR1 comprises KASQDVGTSVA (SEQ ID NO: 20); CDR2            comprises WTSTRHT (SEQ ID NO: 21); and CDR3 comprises            QQYSLYRS (SEQ ID NO: 22);        -   and the CDRs of the heavy chain variable region of said            anti-CEA antibody comprise: CDR1 comprises TYWMS (SEQ ID NO:            23); CDR2 comprises EIHPDSSTINYAPSLKD (SEQ ID NO: 24); and            CDR3 comprises LYFGFPWFAY (SEQ ID NO: 25).    -   US20100221175; US20090092598; US20070202044; US20110064653;        US20090185974; US20080069775.

(42) MET (Met Proto-Oncogene; Hepatocyte Growth Factor Receptor)Nucleotide

Genbank accession no M35073

Genbank version no. M35073.1 GI: 187553

Genbank record update date: Mar. 6, 2012 11:12 AM

Polypeptide

Genbank accession no. AAA59589

Genbank version no. AAA59589.1 GI: 553531

Genbank record update date: Mar. 6, 2012 11:12 AM

Cross References

Dean M., et al Nature 318 (6044), 385-388 (1985)

Other Information

Official Symbol: MET

Other Aliases: AUTS9, HGFR, RCCP2, c-Met

Other Designations: HGF receptor; HGF/SF receptor; SF receptor;hepatocyte growth factor receptor; met proto-oncogene tyrosine kinase;proto-oncogene c-Met; scatter factor receptor; tyrosine-protein kinaseMet

Antibodies

Abgenix/Pfizer: US20100040629

-   -   for example, the antibody produced by hybridoma 13.3.2 having        American Type Culture Collection (ATCC) accession number        PTA-5026; the antibody produced by hybridoma 9.1.2 having ATCC        accession number PTA-5027; the antibody produced by hybridoma        8.70.2 having ATCC accession number PTA-5028; or the antibody        produced by hybridoma 6.90.3 having ATCC accession number        PTA-5029.

Amgen/Pfizer: US20050054019

-   -   for example, an antibody comprising a heavy chain having the        amino acid sequences set forth in SEQ ID NO: 2 where X2 is        glutamate and X4 is serine and a light chain having the amino        acid sequence set forth in SEQ ID NO: 4 where X8 is alanine,        without the signal sequences; an antibody comprising a heavy        chain having the amino acid sequences set forth in SEQ ID NO: 6        and a light chain having the amino acid sequence set forth in        SEQ ID NO: 8, without the signal sequences; an antibody        comprising a heavy chain having the amino acid sequences set        forth in SEQ ID NO: 10 and a light chain having the amino acid        sequence set forth in SEQ ID NO: 12, without the signal        sequences; or an antibody comprising a heavy chain having the        amino acid sequences set forth in SEQ ID NO: 14 and a light        chain having the amino acid sequence set forth in SEQ ID NO: 16,        without the signal sequences.

Agouron Pharmaceuticals (Now Pfizer): US20060035907

Eli Lilly: US20100129369

Genentech: U.S. Pat. No. 5,686,292; US20100028337; US20100016241;US20070129301; US20070098707; US20070092520, US20060270594;US20060134104; US20060035278; US20050233960; US20050037431

-   -   U.S. Pat. No. 5,686,292—for example, ATCC HB-11894 and ATCC        HB-11895    -   US 20100016241—for example, ATCC HB-11894 (hybridoma 1A3.3.13)        or HB-11895 (hybridoma 5D5.11.6)

National Defense Medical Center, Taiwan: Lu R M., et al Biomaterials.2011 April; 32(12):3265-74.

Novartis: US20090175860

-   -   for example, an antibody comprising the sequences of CDR1, CDR2        and CDR3 of heavy chain 4687, wherein the sequences of CDR1,        CDR2, and CDR3 of heavy chain 4687 are residues 26-35, 50-65,        and 98-102, respectively, of SEQ ID NO: 58; and the sequences of        CDR1, CDR2, and CDR3 of light chain 5097, wherein the sequences        of CDR1, CDR2, and CDR3 oflight chain 5097 are residues 24-39,        55-61, and 94-100 of SEQ ID NO: 37.

Pharmacia Corporation: US20040166544

Pierre Fabre: US20110239316, US20110097262, US20100115639

Sumsung: US 20110129481—for example a monoclonal antibody produced froma hybridoma cell having accession number KCLRF-BP-00219 or accessionnumber of KCLRF-BP-00223.

Samsung: US 20110104176—for example an antibody produced by a hybridomacell having Accession Number: KCLRF-BP-00220.

University of Turin Medical School: DN-30 Pacchiana G., et al J BiolChem. 2010 Nov. 12; 285(46):36149-57

Van Andel Research Institute: Jiao Y., et al Mol Biotechnol. 2005September; 31(1):41-54.

(43) MUCI (Mucin 1, Cell Surface Associated) Nucleotide

Genbank accession no J05581

Genbank version no. J05581.1 GI: 188869

Genbank record update date: Jun. 23, 2010 08:48 AM

Polypeptide

Genbank accession no. AAA59876

Genbank version no. AAA59876.1 GI: 188870

Genbank record update date: Jun. 23, 2010 08:48 AM

Cross References

Gendler S. J., et al J. Biol. Chem. 265 (25), 15286-15293 (1990)

Other Information

Official Symbol: MUC1

Other Aliases: RP11-263K19.2, CD227, EMA, H23AG, KL-6, MAM6, MUC-1,MUC-1/SEC, MUC-1/X, MUC1/ZD, PEM, PEMT, PUM

Other Designations: DF3 antigen; H23 antigen; breastcarcinoma-associated antigen DF3; carcinoma-associated mucin; episialin;krebs von den Lungen-6; mucin 1, transmembrane; mucin-1; peanut-reactiveurinary mucin; polymorphic epithelial mucin; tumor associated epithelialmucin; tumor-associated epithelial membrane antigen; tumor-associatedmucin

Antibodies

AltaRex—Quest Pharma Tech: U.S. Pat. No. 6,716,966—for example an Alt-1antibody produced by the hybridoma ATCC No PTA-975.

AltaRex—Quest Pharma Tech: U.S. Pat. No. 7,147,850

CRT: 5E5—Sørensen A L., et al Glycobiology vol. 16 no. 2 pp. 96-107,2006; HMFG2—Burchell J., et al Cancer Res., 47, 5476-5482 (1987); seeWO2015/159076

Glycotope GT-MAB: GT-MAB 2.5-GEX (Website:http://www.glycotope.com/pipeline/pankomab-gex)

Immunogen: U.S. Pat. No. 7,202,346

-   -   for example, antibody MJ-170: hybridoma cell line MJ-170 ATCC        accession no. PTA-5286Monoclonal antibody MJ-171: hybridoma cell        line MJ-171 ATCC accession no. PTA-5287; monoclonal antibody        MJ-172: hybridoma cell line MJ-172 ATCC accession no. PTA-5288;        or monoclonal antibody MJ-173: hybridoma cell line MJ-173 ATCC        accession no. PTA-5302

Immunomedics: U.S. Pat. No. 6,653,104

Ramot Tel Aviv Uni: U.S. Pat. No. 7,897,351

Regents Uni. CA: U.S. Pat. No. 7,183,388; US20040005647; US20030077676.

Roche GlycArt: U.S. Pat. No. 8,021,856

Russian National Cancer Research Center: Imuteran-Ivanov P K., et alBiotechnol J. 2007 July; 2(7):863-70

Technische Univ Braunschweig: (llB6, HT186-B7, HT186-D11, HT186-G2,HT200-3A-C1, HT220-M-D1, HT220-M-G8)—Thie H., et al PLoS One. 2011 Jan.14; 6(1):e15921

(44) CA9 (Carbonic Anhydrase IX) Nucleotide

Genbank accession no. X66839

Genbank version no. X66839.1 GI: 1000701

Genbank record update date: Feb. 2, 2011 10:15 AM

Polypeptide

Genbank accession no. CAA47315

Genbank version no. CAA47315.1 GI: 1000702

Genbank record update date: Feb. 2, 2011 10:15 AM

Cross References

Pastorek J., et al Oncogene 9 (10), 2877-2888 (1994)

Other Information

Official Symbol: CA9

Other Aliases: CAIX, MN

Other Designations: CA-IX; P54/58N; RCC-associated antigen G250;RCC-associated protein G250; carbonate dehydratase IX; carbonicanhydrase 9; carbonic dehydratase; membrane antigen MN; pMW1; renal cellcarcinoma-associated antigen G250

Antibodies

Abgenix/Amgen: US20040018198

Affibody: Anti-CAIX Affibody molecules

-   -   (http://www.affibody.com/en/Product-Portfolio/Pipeline/)

Bayer: U.S. Pat. No. 7,462,696

Bayer/Morphosys: 3ee9 mAb—Petrul H M., et al Mol Cancer Ther. 2012February; 11(2):340-9

Harvard Medical School: Antibodies G10, G36, G37, G39, G45, G57, G106,G119, G6, G27, G40 and G125. Xu C., et al PLoS One. 2010 Mar. 10;5(3):e9625

Institute of Virology, Slovak Academy of Sciences (Bayer)—U.S. Pat. No.5,955,075

-   -   for example, M75—ATCC Accession No. HB 11128 or MN12—ATCC        Accession No. HB 11647

Institute of Virology, Slovak Academy of Sciences: U.S. Pat. No.7,816,493

-   -   for example the M75 monoclonal antibody that is secreted from        the hybridoma VU-M75, which was deposited at the American Type        Culture Collection under ATCC No. HB 11128; or the V/10        monoclonal antibody secreted from the hybridoma V/10-VU, which        was deposited at the International Depository Authority of the        Belgian Coordinated Collection of Microorganisms (BCCM) at the        Laboratorium voor Moleculaire Bioloqie-Plasmidencollectie (LMBP)        at the Universeit Gent in Gent, Belgium, under Accession No.        LMBP 6009CB.

Institute of Virology, Slovak Academy of Sciences US20080177046;US20080176310; US20080176258; US20050031623

Novartis: US20090252738

Wilex: U.S. Pat. No. 7,691,375—for example the antibody produced by thehybridoma cell line DSM ASC 2526.

Wilex: US20110123537; Rencarex: Kennett R H., et al Curr Opin Mol Ther.2003 February; 5(1):70-5

Xencor: US20090162382

(45) EGFRvIII (Epidermal Growth Factor Receptor (EGFR), TranscriptVariant 3, Nucleotide

Genbank accession no. NM_201283

Genbank version no. NM_201283.1 GI: 41327733

Genbank record update date: Sep. 30, 2012 01:47 PM

Polypeptide

Genbank accession no. NP_958440

Genbank version no. NP_958440.1 GI: 41327734

Genbank record update date: Sep. 30, 2012 01:47 PM

Cross-References

Batra S K., et al Cell Growth Differ 1995; 6:1251-1259.

Antibodies:

U.S. Pat. Nos. 7,628,986 and 7,736,644 (Amgen)

-   -   For example, a heavy chain variable region amino acid sequence        selected from the group consisting of SEQ ID NO: 142 and        variants & a light chain variable region amino acid sequence        selected from the group consisting of: SEQ ID NO: 144 and        variants.

US20100111979 (Amgen)

-   -   For example, an antibody comprising a heavy chain amino acid        sequence comprising:    -   CDR1 consisting of a sequence selected from the group consisting        of the amino acid sequences for the CDR1 region of antibodies        13.1.2 (SEQ ID NO: 138), 131 (SEQ ID NO: 2), 170 (SEQ ID NO: 4),        150 (SEQ ID NO: 5), 095 (SEQ ID NO: 7), 250 (SEQ ID NO: 9), 139        (SEQ ID NO: 10), 211 (SEQ ID NO: 12), 124 (SEQ ID NO: 13), 318        (SEQ ID NO: 15), 342 (SEQ ID NO: 16), and 333 (SEQ ID NO: 17);    -   CDR2 consisting of a sequence selected from the group consisting        of the amino acid sequences for the CDR2 region of antibodies        13.1.2 (SEQ ID NO: 138), 131 (SEQ ID NO: 2), 170 (SEQ ID NO: 4),        150 (SEQ ID NO: 5), 095 (SEQ ID NO: 7), 250 (SEQ ID NO: 9), 139        (SEQ ID NO: 10), 211 (SEQ ID NO: 12), 124 (SEQ ID NO: 13), 318        (SEQ ID NO: 15), 342 (SEQ ID NO: 16), and 333 (SEQ ID NO: 17);        and    -   CDR3 consisting of a sequence selected from the group consisting        of the amino acid sequences for the CDR3 region of antibodies        13.1.2 (SEQ ID NO: 138), 131 (SEQ ID NO: 2), 170 (SEQ ID NO: 4),        150 (SEQ ID NO: 5), 095 (SEQ ID NO: 7), 250 (SEQ ID NO: 9), 139        (SEQ ID NO: 10), 211 (SEQ ID NO: 12), 124 (SEQ ID NO: 13), 318        (SEQ ID NO: 15), 342 (SEQ ID NO: 16), and 333 (SEQ ID NO: 17).

US20090240038 (Amgen)

-   -   For example, an antibody having at least one of the heavy or        light chain polypeptides comprises an amino acid sequence that        is at least 90% identical to the amino acid sequence selected        from the group consisting of: SEQ ID NO: 2, SEQ ID NO: 19, SEQ        ID NO: 142, SEQ ID NO: 144, and any combination thereof.

US20090175887 (Amgen)

-   -   For example, an antibody having a heavy chain amino acid        sequence selected from the group consisting of the heavy chain        amino acid sequence of antibody 13.1.2 (SEQ ID NO: 138), 131        (SEQ ID NO: 2), 170 (SEQ ID NO: 4), 150 (SEQ ID NO: 5), 095 (SEQ        ID NO: 7), 250 (SEQ ID NO: 9), 139 (SEQ ID NO: 10), 211 (SEQ ID        NO: 12), 124 (SEQ ID NO: 13), 318 (SEQ ID NO: 15), 342 (SEQ ID        NO: 16), and 333 (SEQ ID NO: 17).

US20090156790 (Amgen)

-   -   For example, antibody having heavy chain polypeptide and a light        chain polypeptide, wherein at least one of the heavy or light        chain polypeptides comprises an amino acid sequence that is at        least 90% identical to the amino acid sequence selected from the        group consisting of: SEQ ID NO: 2, SEQ ID NO: 19, SEQ ID NO:        142, SEQ ID NO: 144, and any combination thereof.

US20090155282, US20050059087 and US20050053608 (Amgen)

-   -   For example, an antibody heavy chain amino acid sequence        selected from the group consisting of the heavy chain amino acid        sequence of antibody 13.1.2 (SEQ ID NO: 138), 131 (SEQ ID NO:        2), 170 (SEQ ID NO: 4), 150 (SEQ ID NO: 5), 095 (SEQ ID NO: 7),        250 (SEQ ID NO: 9), 139 (SEQ ID NO: 10), 211 (SEQ ID NO: 12),        124 (SEQ ID NO: 13), 318 (SEQ ID NO: 15), 342 (SEQ ID NO: 16),        and 333 (SEQ ID NO: 17).

MR1-1 (U.S. Pat. No. 7,129,332; Duke)

-   -   For example, a variant antibody having the sequence of SEQ ID        NO.18 with the substitutions S98P-T99Y in the CDR3 VH, and F92W        in CDR3 VL.

L8A4, H10, Y10 (Wikstrand C J., et al Cancer Res. 1995 Jul. 15;55(14):3140-8; Duke)

US20090311803 (Harvard University)

-   -   For example, SEQ ID NO:9 for antibody heavy chain variable        region, and SEQ ID NO: 3 for light chain variable region amino        acid sequences

US20070274991 (EMD72000, also known as matuzumab; Harvard University)

-   -   For example, SEQ ID NOs: 3 & 9 for light chain and heavy chain        respectively

U.S. Pat. No. 6,129,915 (Schering)

-   -   For example, SEQ. ID NOs: 1, 2, 3, 4, 5 and 6.

mAb CH12—Wang H., et al FASEB J. 2012 January; 26(1):73-80 (ShanghaiCancer Institute).

RAbDMvIII—Gupta P., et al BMC Biotechnol. 2010 Oct. 7; 10:72 (StanfordUniversity Medical Center).

mAb Ua30—Ohman L., et al Tumour Biol. 2002 March-April; 23(2):61-9(Uppsala University).

Han D G., et al Nan Fang Yi Ke Da Xue Xue Bao. 2010 January; 30(1):25-9(Xi'an Jiaotong University).

(46) CD33 (CD33 Molecule) Nucleotide

Genbank accession no. M_23197

Genbank version no. NM_23197.1 GI: 180097

Genbank record update date: Jun. 23, 2010 08:47 AM

Polypeptide

Genbank accession no. AAA51948

Genbank version no. AAA51948.1 GI: 188098

Genbank record update date: Jun. 23, 2010 08:47 AM

Cross-References

Simmons D., et al J. Immunol. 141 (8), 2797-2800 (1988)

Other Information

Official Symbol: CD33

Other Aliases: SIGLEC-3, SIGLEC3, p67

Other Designations: CD33 antigen (gp67); gp67; myeloid cell surfaceantigen CD33; sialic acid binding Ig-like lectin 3; sialic acid-bindingIg-like lectin

Antibodies

H195 (Lintuzumab)—Raza A., et al Leuk Lymphoma. 2009 August;50(8):1336-44; U.S. Pat. No. 6,759,045 (Seattle Genetics/Immunomedics)

mAb OKT9: Sutherland, D. R. et al. Proc Natil Acad Sci USA 78(7):4515-4519 1981, Schneider, C., et al J Biol Chem 257, 8516-8522 (1982)

mAb E6: Hoogenboom, H. R., et al J Immunol 144, 3211-3217 (1990)

U.S. Pat. No. 6,590,088 (Human Genome Sciences)

-   -   For example, SEQ ID NOs: 1 and 2 and ATCC accession no. 97521

U.S. Pat. No. 7,557,189 (Immunogen)

-   -   For example, an antibody or fragment thereof comprising a heavy        chain variable region which comprises three CDRs having the        amino acid sequences of SEQ ID NOs:1-3 and a light chain        variable region comprising three CDRs having the amino acid        sequences of SEQ ID NOs:4-6.

(47) CD19 (CD19 Molecule) Nucleotide

Genbank accession no. NM_001178098

Genbank version no. NM_001178098.1 GI: 296010920

Genbank record update date: Sep. 10, 2012 12:43 AM

Polypeptide

Genbank accession no. NP_001171569

Genbank version no. NP_001171569.1 GI: 296010921

Genbank record update date: Sep. 10, 2012 12:43 AM

Cross-References

Tedder T F., et al J. Immunol. 143 (2): 712-7 (1989)

Other Information

Official Symbol: CD19

Other Aliases: B4, CVID3

Other Designations: B-lymphocyte antigen CD19; B-lymphocyte surfaceantigen B4; T-cell surface antigen Leu-12; differentiation antigen CD19

Antibodies

Immunogen: HuB4—Al-Katib A M., et al Clin Cancer Res. 2009 Jun. 15;15(12):4038-45.

4G7: Kügler M., et al Protein Eng Des Sel. 2009 March; 22(3):135-47

-   -   For example, sequences in FIG. 3 of of Knappik, A. et al. J Mol        Biol 2000 February; 296(1):57-86

AstraZeneca/MedImmune: MEDI-551—Herbst R., et al J Pharmacol Exp Ther.2010 October; 335(1):213-22

Glenmark Pharmaceuticals: GBR-401—Hou S., et al Mol Cancer Ther November2011 (Meeting Abstract Supplement) C164

U.S. Pat. No. 7,109,304 (Immunomedics)

-   -   For example, an antibody comprising the sequence of hA19Vk (SEQ        ID NO:7) and the sequence of hA19VH (SEQ ID NO:10)

U.S. Pat. No. 7,902,338 (Immunomedics)

-   -   For example, an antibody or antigen-binding fragment thereof        that comprises the light chain complementarity determining        region CDR sequences CDR1 of SEQ ID NO: 16 (KASQSVDYDGDSYLN);        CDR2 of SEQ ID NO: 17 (DASNLVS); and CDR3 of SEQ ID NO: 18        (QQSTEDPWT) and the heavy chain CDR sequences CDR1 of SEQ ID NO:        19 (SYWMN); CDR2 of SEQ ID NO: 20 (QIWPGDGDTNYNGKFKG) and CDR3        of SEQ ID NO: 21 (RETTTVGRYYYAMDY) and also comprises human        antibody framework (FR) and constant region sequences with one        or more framework region amino acid residues substituted from        the corresponding framework region sequences of the parent        murine antibody, and wherein said substituted FR residues        comprise the substitution of serine for phenylalanine at Kabat        residue 91 of the heavy chain variable region.

Medarex: MDX-1342—Cardarelli P M., et al Cancer Immunol Immunother. 2010February; 59(2):257-65.

MorphoSys/Xencor: MOR-208/XmAb-5574—Zalevsky J., et al Blood. 2009 Apr.16; 113(16):3735-43

U.S. Pat. No. 7,968,687 (Seattle Genetics)

-   -   An antibody or antigen-binding fragment comprising a heavy chain        variable domain comprising the amino acid sequence of SEQ ID        NO:9 and a light chain variable domain comprising the amino acid        sequence of SEQ ID NO: 24.

4G7 chim—Lang P., et al Blood. 2004 May 15; 103(10):3982-5 (Universityof Tübingen)

-   -   For example, FIG. 6 and SEQ ID No: 80 of US20120082664

Zhejiang University School of Medicine: 2E8—Zhang J., et al J DrugTarget. 2010 November; 18(9):675-8

(48) IL2RA (Interleukin 2 Receptor, Alpha); NCBI Reference Sequence:NM_000417.2); Nucleotide

Genbank accession no. NM_000417

Genbank version no. NM_000417.2 GI: 269973860

Genbank record update date: Sep. 9, 2012 04:59 PM

Polypeptide

Genbank accession no. NP_000408

Genbank version no. NP_000408.1 GI: 4557667

Genbank record update date: Sep. 9, 2012 04:59 PM

Cross-References

Kuziel W. A., et al J. Invest. Dermatol. 94 (6 SUPPL), 27S-32S (1990)

Other Information

Official Symbol: IL2RA

Other Aliases: RP11-536K7.1, CD25, IDDM10, IL2R, TCGFR

Other Designations: FIL-2 receptor subunit alpha; IL-2-RA; IL-2R subunitalpha; IL2-RA; TAC antigen; interleukin-2 receptor subunit alpha; p55

Antibodies

U.S. Pat. No. 6,383,487 (Novartis/UCL: Baxilisimab [Simulect])

U.S. Pat. No. 6,521,230 (Novartis/UCL: Baxilisimab [Simulect])

-   -   For example, an antibody having an antigen binding site        comprises at least one domain which comprises CDR1 having the        amino acid sequence in SEQ. ID. NO: 7, CDR2 having the amino        acid sequence in SEQ. ID. NO: 8, and CDR3 having the amino acid        sequence in SEQ. ID. NO: 9; or said CDR1, CDR2 and CDR3 taken in        sequence as a whole comprise an amino acid sequence which is at        least 90% identical to SEQ. ID. NOs: 7, 8 and 9 taken in        sequence as a whole.

Daclizumab—Rech A J., et al Ann N YAcad Sci. 2009 September; 1174:99-106(Roche)

(49) AXL (AXL Receptor Tyrosine Kinase) Nucleotide

Genbank accession no. M76125

Genbank version no. M76125.1 GI: 292869

Genbank record update date: Jun. 23, 2010 08:53 AM

Polypeptide

Genbank accession no. AAA61243

Genbank version no. AAA61243.1 GI: 29870

Genbank record update date: Jun. 23, 2010 08:53 AM

Cross-References

O'Bryan J. P., et al Mol. Cell. Biol. 11 (10), 5016-5031 (1991);Bergsagel P. L., et al J. Immunol. 148 (2), 590-596 (1992)

Other Information

Official Symbol: AXL

Other Aliases: JTK11, UFO

Other Designations: AXL oncogene; AXL transforming sequence/gene;oncogene AXL; tyrosine-protein kinase receptor UFO

Antibodies

YW327.6S2—Ye X., et al Oncogene. 2010 Sep. 23; 29(38):5254-64.(Genentech)

BergenBio: BGB324 (http://www.bergenbio.com/BGB324)

(50) CD30—TNFRSF8 (Tumor Necrosis Factor Receptor Superfamily, Member 8)Nucleotide

Genbank accession no. M83554

Genbank version no. M83554.1 GI: 180095

Genbank record update date: Jun. 23, 2010 08:53 AM

Polypeptide

Genbank accession no. AAA51947

Genbank version no. AAA51947.1 GI: 180096

Genbank record update date: Jun. 23, 2010 08:53 AM

Cross-References

Durkop H., et al Cell 68 (3), 421-427 (1992)

Other Information

Official Symbol: TNFRSF8

Other Aliases: CD30, D1S166E, Ki-1

Other Designations: CD30L receptor; Ki-1 antigen; cytokine receptorCD30; lymphocyte activation antigen CD30; tumor necrosis factor receptorsuperfamily member 8

(51) BCMA (B-cell maturation antigen)—TNFRSF17 (Tumor necrosis factorreceptor superfamily, member 17)

Nucleotide

Genbank accession no. Z29574

Genbank version no. Z29574.1 GI: 471244

Genbank record update date: Feb. 2, 2011 10:40 AM

Polypeptide

Genbank accession no. CAA82690

Genbank version no. CAA82690.1 GI: 471245

Genbank record update date: Feb. 2, 2011 10:40 AM

Cross-References

Laabi Y., et al Nucleic Acids Res. 22 (7), 1147-1154 (1994)

Other Information

Official Symbol: TNFRSF17

Other Aliases: BCM, BCMA, CD269

Other Designations: B cell maturation antigen; B-cell maturation factor;B-cell maturation protein; tumor necrosis factor receptor superfamilymember 17

(52) CT Ags—CTA (Cancer Testis Antigens) Cross-References

Fratta E., et al. Mol Oncol. 2011 April; 5(2):164-82; Lim S H., at al AmJ Blood Res. 2012; 2(1):29-35.

(53) CD174 (Lewis Y)—FUT3 (fucosyltransferase 3 (Galactoside3(4)-L-fucosyltransferase, Lewis Blood Group)

Nucleotide

Genbank accession no. NM000149

Genbank version no. NM000149.3 GI: 148277008

Genbank record update date: Jun. 26, 2012 04:49 PM

Polypeptide

Genbank accession no. NP_000140

Genbank version no. NP_000140.1 GI: 4503809

Genbank record update date: Jun. 26, 2012 04:49 PM

Cross-References

Kukowska-Latallo, J. F., et al Genes Dev. 4 (8), 1288-1303 (1990)

Other Information

Official Symbol: FUT3

Other Aliases: CD174, FT3B, FucT-III, LE, Les

Other Designations: Lewis FT; alpha-(1,3/1,4)-fucosyltransferase; bloodgroup Lewis alpha-4-fucosyltransferase; fucosyltransferase III;galactoside 3(4)-L-fucosyltransferase

(54) CLEC14A (C-Type Lectin Domain Family 14, Member a; GenbankAccession No. NM175060)

Nucleotide

Genbank accession no. NM175060

Genbank version no. NM175060.2 GI: 371123930

Genbank record update date: Apr. 1, 2012 03:34 PM

Polypeptide

Genbank accession no. NP_778230

Genbank version no. NP_778230.1 GI: 28269707

Genbank record update date: Apr. 1, 2012 03:34 PM

Other Information

Official Symbol: CLEC14A

Other Aliases: UNQ236/PRO269, C14orf27, CEG1, EGFR-5

Other Designations: C-type lectin domain family 14 member A; CIECT andEGF-like domain containing protein; epidermal growth factor receptor 5

(55) GRP78—HSPA5 (heat shock 70 kDa protein 5 (glucose-regulatedprotein, 78 kDa)

Nucleotide

Genbank accession no. NM005347

Genbank version no. NM005347.4 GI: 305855105

Genbank record update date: Sep. 30, 2012 01:42 PM

Polypeptide

Genbank accession no. NP_005338

Genbank version no. NP_005338.1 GI: 16507237

Genbank record update date: Sep. 30, 2012 01:42 PM

Cross-References

Ting J., et al DNA 7 (4), 275-286 (1988)

Other Information

Official Symbol: HSPA5

Other Aliases: BIP, GRP78, MIF2

Other Designations: 78 kDa glucose-regulated protein; endoplasmicreticulum lumenal Ca(2+)-binding protein grp78; immunoglobulin heavychain-binding protein

(56) Cd70 (Cd70 Molecule) L08096 Nucleotide

Genbank accession no. L08096

Genbank version no. L08096.1 GI: 307127

Genbank record update date: Jun. 23, 2012 08:54 AM

Polypeptide

Genbank accession no. AAA36175

Genbank version no. AAA36175.1 GI: 307128

Genbank record update date: Jun. 23, 2012 08:54 AM

Cross-References

Goodwin R. G., et al Cell 73 (3), 447-456 (1993)

Other Information

Official Symbol: CD70

Other Aliases: CD27L, CD27LG, TNFSF7

Other Designations: CD27 ligand; CD27-L; CD70 antigen; Ki-24 antigen;surface antigen CD70; tumor necrosis factor (ligand) superfamily, member7; tumor necrosis factor ligand superfamily member 7

Antibodies

MDX-1411 against CD70 (Medarex)

h1F6 (Oflazoglu, E., et al, Clin Cancer Res. 2008 Oct. 1;14(19):6171-80; Seattle Genetics)

-   -   For example, see US20060083736 SEQ ID NOs: 1, 2, 11 and 12 and        FIG. 1.        (57) Stem Cell Specific Antigens. For Example:    -   5T4 (see entry (63) below)    -   CD25 (see entry (48) above)    -   CD32        -   Polypeptide            -   Genbank accession no. ABK42161            -   Genbank version no. ABK42161.1 GI: 117616286            -   Genbank record update date: Jul. 25, 2007 03:00 PM            -   LGR5/GPR49        -   Nucleotide            -   Genbank accession no. NM_003667            -   Genbank version no. NM_003667.2 GI: 24475886            -   Genbank record update date: Jul. 22, 2012 03:38 PM        -   Polypeptide            -   Genbank accession no. NP_003658            -   Genbank version no. NP_003658.1 GI: 4504379            -   Genbank record update date: Jul. 22, 2012 03:38 PM    -   Prominin/CD133        -   Nucleotide            -   Genbank accession no. NM_006017            -   Genbank version no. NM_006017.2 GI: 224994187            -   Genbank record update date: Sep. 30, 2012 01:47 PM        -   Polypeptide            -   Genbank accession no. NP_006008            -   Genbank version no. NP_006008.1 GI: 5174387            -   Genbank record update date: Sep. 30, 2012 01:47 PM

(58) ASG-5 Cross-References

(Smith L. M., et. al AACR 2010 Annual Meeting (abstract #2590); Gudas J.M., et. al. AACR 2010 Annual Meeting (abstract #4393)

Antibodies

Anti-AGS-5 Antibody: M6.131 (Smith, L. M., et. al AACR 2010 AnnualMeeting (abstract #2590)

(59) ENPP3 (Ectonucleotide Pyrophosphatase/Phosphodiesterase 3)Nucleotide

Genbank accession no. AF005632

Genbank version no. AF005632.2 GI: 4432589

Genbank record update date: Mar. 10, 2010 09:41 PM

Polypeptide

Genbank accession no. AAC51813

Genbank version no. AAC51813.1 GI: 2465540

Genbank record update date: Mar. 10, 2010 09:41 PM

Cross-References

Jin-Hua P., et al Genomics 45 (2), 412-415 (1997)

Other Information

Official Symbol: ENPP3

Other Aliases: RP5-988G15.3, B10, CD203c, NPP3, PD-IBETA, PDNP3

Other Designations: E-NPP 3; dJ1005H11.3 (phosphodiesterase 1/nucleotidepyrophosphatase 3); dJ914N13.3 (phosphodiesterase 1/nucleotidepyrophosphatase 3); ectonucleotide pyrophosphatase/phosphodiesterasefamily member 3; gp130RB13-6; phosphodiesterase I beta;phosphodiesterase I/nucleotide pyrophosphatase 3; phosphodiesterase-Ibeta

(60) PRR4 (Proline Rich 4 (Lacrimal)) Nucleotide

Genbank accession no. NM_007244

Genbank version no. NM_007244.2 GI: 154448885

Genbank record update date: Jun. 28, 2012 12:39 PM

Polypeptide

Genbank accession no. NP_009175

Genbank version no. NP_009175.2 GI: 154448886

Genbank record update date: Jun. 28, 2012 12:39 PM

Cross-References

Dickinson D. P., et al Invest. Ophthalmol. Vis. Sci. 36 (10), 2020-2031(1995)

Other Information

Official Symbol: PRR4

Other Aliases: LPRP, PROL4

Other Designations: lacrimal proline-rich protein; nasopharyngealcarcinoma-associated proline-rich protein 4; proline-rich polypeptide 4;proline-rich protein 4

(61) GCC—GUCY2C (guanylate cyclase 2C (heat stable enterotoxin receptor)

Nucleotide

Genbank accession no. NM_004963

Genbank version no. NM_004963.3 GI: 222080082

Genbank record update date: Sep. 2, 2012 01:50 PM

Polypeptide

Genbank accession no. NP_004954

Genbank version no. NP_004954.2 GI: 222080083

Genbank record update date: Sep. 2, 2012 01:50 PM

Cross-References

De Sauvage F. J., et al J. Biol. Chem. 266 (27), 17912-17918 (1991);Singh S., et al Biochem. Biophys. Res. Commun. 179 (3), 1455-1463 (1991)

Other Information

Official Symbol: GUCY2C

Other Aliases: DIAR6, GUC2C, MUCIL, STAR

Other Designations: GC-C; STA receptor; guanylyl cyclase C; hSTAR;heat-stable enterotoxin receptor; intestinal guanylate cyclase

(62) Liv-1—SLC39A6 (Solute Carrier Family 39 (Zinc Transporter), Member6) Nucleotide

Genbank accession no. U41060

Genbank version no. U41060.2 GI: 12711792

Genbank record update date: Nov. 30, 2009 04:35 PM

Polypeptide

Genbank accession no. AAA96258

Genbank version no. AAA96258.2 GI: 12711793

Genbank record update date: Nov. 30, 2009 04:35 PM

Cross-References

Taylor K M., et al Biochim Biophys Acta. 2003 Apr. 1; 1611(1-2):16-30

Other Information

Official Symbol: SLC39A6

Other Aliases: LIV-1

Other Designations: LIV-1 protein, estrogen regulated; ZIP-6;estrogen-regulated protein LIV-1; solute carrier family 39 (metal iontransporter), member 6; solute carrier family 39 member 6; zinctransporter ZIP6; zrt- and lrt-like protein 6

(63) 5T4, Trophoblast glycoprotein, TPBG—TPBG (trophoblast glycoprotein)

Nucleotide

Genbank accession no. AJ012159

Genbank version no. AJ012159.1 GI: 3805946

Genbank record update date: Feb. 1, 2011 10:27 AM

Polypeptide

Genbank accession no. CAA09930

Genbank version no. CAA09930.1 GI: 3805947

Genbank record update date: Feb. 1, 2011 10:27 AM

Cross-References

King K. W., et al Biochim. Biophys. Acta 1445 (3), 257-270 (1999)

Other Information

-   -   Official Symbol: TPBG    -   Other Aliases: 5T4, 5T4AG, M6P1    -   Other Designations: 5T4 oncofetal antigen; 5T4 oncofetal        trophoblast glycoprotein; 5T4 oncotrophoblast glycoprotein    -   See WO2015/155345

(64) CD56—NCMA1 (Neural Cell Adhesion Molecule 1) Nucleotide

Genbank accession no. NM_000615

Genbank version no. NM_000615.6 GI: 336285433

Genbank record update date: Sep. 23, 2012 02:32 PM

Polypeptide

Genbank accession no. NP_000606

Genbank version no. NP_000606.3 GI: 94420689

Genbank record update date: Sep. 23, 2012 02:32 PM

Cross-References

Dickson, G., et al, Cell 50 (7), 1119-1130 (1987)

Other Information

Official Symbol: NCAM1

Other Aliases: CD56, MSK39, NCAM

Other Designations: antigen recognized by monoclonal antibody 5.1H11;neural cell adhesion molecule, NCAM

Antibodies

Immunogen: HuN901 (Smith S V., et al Curr Opin Mol Ther. 2005 August;7(4):394-401)

-   -   For example, see humanized from murine N901 antibody. See FIGS.        1b and 1e of Roguska, M. A., et al. Proc Natl Acad Sci USA        February 1994; 91:969-973.

(65) CanAg (Tumor Associated Antigen CA242) Cross-References

Haglund C., et al Br J Cancer 60:845-851, 1989; Baeckstrom D., et al JBiol Chem 266:21537-21547, 1991

Antibodies

huC242 (Tolcher A W et al., J Clin Oncol. 2003 Jan. 15; 21(2):211-22;Immunogen)

-   -   For example, see US20080138898A1 SEQ ID NO: 1 and 2

(66) FOLR1 (Folate Receptor 1) Nucleotide

Genbank accession no. J05013

Genbank version no. J05013.1 GI: 182417

Genbank record update date: Jun. 23, 2010 08:47 AM

Polypeptide

Genbank accession no. AAA35823

Genbank version no. AAA35823.1 GI: 182418

Genbank record update date: Jun. 23, 2010 08:47 AM

Cross-References

Elwood P. C., et al J. Biol. Chem. 264 (25), 14893-14901 (1989)

Other Information

Official Symbol: FOLR1

Other Aliases: FBP, FOLR

Other Designations: FR-alpha; KB cells FBP; adult folate-bindingprotein; folate binding protein; folate receptor alpha; folate receptor,adult; ovarian tumor-associated antigen MOv18

Antibodies

M9346A—Whiteman K R., et al Cancer Res Apr. 15, 2012; 72(8 Supplement):4628 (Immunogen)

(67) GPNMB (Glycoprotein (transmembrane) nmb)

Nucleotide

Genbank accession no. X76534

Genbank version no. X76534.1 GI: 666042

Genbank record update date: Feb. 2, 2011 10:10 AM

Polypeptide

Genbank accession no. CAA54044

Genbank version no. CAA54044.1 GI: 666043

Genbank record update date: Feb. 2, 2011 10:10 AM

Cross-References

Weterman M. A., et al Int. J. Cancer 60 (1), 73-81 (1995)

Other Information

Official Symbol: GPNMB

Other Aliases: UNQ1725/PRO9925, HGFIN, NMB

Other Designations: glycoprotein NMB; glycoprotein nmb-like protein;osteoactivin; transmembrane glycoprotein HGFIN; transmembraneglycoprotein NMB

Antibodies

Celldex Therapeutics: CR011 (Tse K F., et al Clin Cancer Res. 2006 Feb.15; 12(4):1373-82)

-   -   For example, see EP1827492B1 SEQ ID NO: 22, 24, 26, 31, 33 and        35

(68) TIM-1—HAVCR1 (Hepatitis a Virus Cellular Receptor 1) Nucleotide

Genbank accession no. AF043724

Genbank version no. AF043724.1 GI: 2827453

Genbank record update date: Mar. 10, 2010 06:24 PM

Polypeptide

Genbank accession no. AAC39862

Genbank version no. AAC39862.1 GI: 2827454

Genbank record update date: Mar. 10, 2010 06:24 PM

Cross-References

Feigelstock D., et al J. Virol. 72 (8), 6621-6628 (1998)

Other Information

Official Symbol: HAVCR1

Other Aliases: HAVCR, HAVCR-1, KIM-1, KIM1, TIM, TIM-1, TIM1, TIMD-1,TIMD1

Other Designations: T cell immunoglobin domain and mucin domain protein1; T-cell membrane protein 1; kidney injury molecule 1

(69) RG-1/Prostate Tumor Target Mindin—Mindin/RG-1 Cross-References

Parry R., et al Cancer Res. 2005 Sep. 15; 65(18):8397-405

(70) B7-H4—VTCNI (V-Set Domain Containing T Cell Activation Inhibitor 1Nucleotide

Genbank accession no. BX648021

Genbank version no. BX648021.1 GI: 34367180

Genbank record update date: Feb. 2, 2011 08:40 AM

Cross-References

Sica G L., et al Immunity. 2003 June; 18(6):849-61

Other Information

Official Symbol: VTCN1

Other Aliases: RP11-229A19.4, B7-H4, B7H4, B7S1, B7X, B7h.5, PRO1291,VCTN1

Other Designations: B7 family member, H4; B7 superfamily member 1; Tcell costimulatory molecule B7x; T-cell costimulatory molecule B7x;V-set domain-containing T-cell activation inhibitor 1; immunecostimulatory protein B7-H4

(71) PTK7 (PTK7 Protein Tyrosine Kinase 7) Nucleotide

Genbank accession no. AF447176

Genbank version no. AF447176.1 GI: 17432420

Genbank record update date: Nov. 28, 2008 01:51 PM

Polypeptide

Genbank accession no. AAL39062

Genbank version no. AAL39062.1 GI: 17432421

Genbank record update date: Nov. 28, 2008 01:51 PM

Cross-References

Park S. K., et al J. Biochem. 119 (2), 235-239 (1996)

Other Information

Official Symbol: PTK7

Other Aliases: CCK-4, CCK4

Other Designations: colon carcinoma kinase 4; inactive tyrosine-proteinkinase 7; pseudo tyrosine kinase receptor 7; tyrosine-proteinkinase-like 7

(72) CD37 (CD37 molecule)

Nucleotide

Genbank accession no. NM_001040031

Genbank version no. NM_001040031.1 GI: 91807109

Genbank record update date: Jul. 29, 2012 02:08 PM

Polypeptide

Genbank accession no. NP_001035120

Genbank version no. NP_001035120.1 GI: 91807110

Genbank record update date: Jul. 29, 2012 02:08 PM

Cross-References

Schwartz-Albiez R., et al J. Immunol. 140 (3), 905-914 (1988)

Other Information

Official Symbol: CD37

Other Aliases: GP52-40, TSPAN26

Other Designations: CD37 antigen; cell differentiation antigen 37;leukocyte antigen CD37; leukocyte surface antigen CD37; tetraspanin-26;tspan-26

Antibodies

Boehringer Ingelheim: mAb 37.1 (Heider K H., et al Blood. 2011 Oct. 13;118(15):4159-68)

Trubion: CD37-SMIP (G28-1 scFv-lg) ((Zhao X., et al Blood. 2007; 110:2569-2577)

-   -   For example, see US20110171208A1 SEQ ID NO: 253

Immunogen: K7153A (Deckert J., et al Cancer Res Apr. 15, 2012; 72(8Supplement): 4625)

(73) CD138—SDC1 (Syndecan 1) Nucleotide

Genbank accession no. AJ551176

Genbank version no. AJ551176.1 GI: 29243141

Genbank record update date: Feb. 1, 2011 12:09 PM

Polypeptide

Genbank accession no. CAD80245

Genbank version no. CAD80245.1 GI: 29243142

Genbank record update date: Feb. 1, 2011 12:09 PM

Cross-References

O'Connell F P., et al Am J Clin Pathol. 2004 February; 121(2):254-63

Other Information

Official Symbol: SDC1

Other Aliases: CD138, SDC, SYND1, syndecan

Other Designations: CD138 antigen; heparan sulfate proteoglycanfibroblast growth factor receptor; syndecan proteoglycan 1; syndecan-1

Antibodies

Biotest: chimerized MAb (nBT062)—(Jagannath S., et al Poster ASH #3060,2010; WIPO Patent Application WO/2010/128087)

-   -   For example, see US20090232810 SEQ ID NO: 1 and 2

Immunogen: B-B4 (Tassone P., et al Blood 104_3688-3696)

-   -   For example, see US20090175863A1 SEQ ID NO: 1 and 2        (74) CD74 (CD74 molecule, major histocompatibility complex,        class II invariant chain)

Nucleotide

Genbank accession no. NM_004355

Genbank version no. NM_004355.1 GI: 343403784

Genbank record update date: Sep. 23, 2012 02:30 PM

Polypeptide

Genbank accession no. NP_004346

Genbank version no. NP_004346.1 GI: 10835071

Genbank record update date: Sep. 23, 2012 02:30 PM

Cross-References

Kudo, J., et al Nucleic Acids Res. 13 (24), 8827-8841 (1985)

Other Information

Official Symbol: CD74

Other Aliases: DHLAG, HLADG, II, Ia-GAMMA

Other Designations: CD74 antigen (invariant polypeptide of majorhistocompatibility complex, class II antigen-associated); HLA class IIhistocompatibility antigen gamma chain; HLA-DR antigens-associatedinvariant chain; HLA-DR-gamma; Ia-associated invariant chain; MHC HLA-DRgamma chain; gamma chain of class II antigens; p33

Antibodies

Immunomedics: hLL1 (Milatuzumab)—Berkova Z., et al Expert Opin InvestigDrugs. 2010 January; 19(1):141-9)

-   -   For example, see US20040115193 SEQ ID NOs: 19, 20, 21, 22, 23        and 24

Genmab: HuMax-CD74 (see website)

(75) Claudins—CLs (Claudins) Cross-References

Offner S., et al Cancer Immunol Immunother. 2005 May; 54(5):431-45,Suzuki H., et al Ann N Y Acad Sci. 2012 July; 1258:65-70)

In humans, 24 members of the family have been described—see literaturereference.

(76) EGFR (Epidermal Growth Factor Receptor) Nucleotide

Genbank accession no. NM_005228

Genbank version no. NM_005228.3 GI: 41927737

Genbank record update date: Sep. 30, 2012 01:47 PM

Polypeptide

Genbank accession no. NP_005219

Genbank version no. NP_005219.2 GI: 29725609

Genbank record update date: Sep. 30, 2012 01:47 PM

Cross-References

Dhomen N S., et al Crit Rev Oncog. 2012; 17(1):31-50

Other Information

Official Symbol: EGFR

Other Aliases: ERBB, ERBB1, HER1, PIG61, mENA

Other Designations: avian erythroblastic leukemia viral (v-erb-b)oncogene homolog; cell growth inhibiting protein 40; cellproliferation-inducing protein 61; proto-oncogene c-ErbB-1; receptortyrosine-protein kinase erbB-1

Antibodies

BMS: Cetuximab (Erbitux)—Broadbridge V T., et al Expert Rev AnticancerTher. 2012 May; 12(5):555-65.

-   -   For example, see U.S. Pat. No. 6,217,866—ATTC deposit No. 9764.

Amgen: Panitumumab (Vectibix)—Argiles G., et al Future Oncol. 2012April; 8(4):373-89

-   -   For example, see U.S. Pat. No. 6,235,883 SEQ ID NOs: 23-38.

Genmab: Zalutumumab—Rivera F., et al Expert Opin Biol Ther. 2009 May;9(5):667-74.

YM Biosciences: Nimotuzumab—Ramakrishnan M S., et al MAbs. 2009January-February; 1(1):41-8.

-   -   For example, see U.S. Pat. No. 5,891,996 SEQ ID NOs: 27-34.        (77) Her3 (ErbB3)—ERBB3 (v-erb-b2 Erythroblastic Leukemia Viral        Oncogene Homolog 3 (Avian))

Nucleotide

Genbank accession no. M34309

Genbank version no. M34309.1 GI: 183990

Genbank record update date: Jun. 23, 2010 08:47 PM

Polypeptide

Genbank accession no. AAA35979

Genbank version no. AAA35979.1 GI: 306841

Genbank record update date: Jun. 23, 2010 08:47 PM

Cross-References

Plowman, G. D., et al., Proc. Natl. Acad. Sci. U.S.A. 87 (13), 4905-4909(1990)

Other Information

Official Symbol: ERBB3

Other Aliases: ErbB-3, HER3, LCCS2, MDA-BF-1, c-erbB-3, c-erbB3,erbB3-S, p180-ErbB3, p45-sErbB3, p85-sErbB3

Other Designations: proto-oncogene-like protein c-ErbB-3; receptortyrosine-protein kinase erbB-3; tyrosine kinase-type cell surfacereceptor HER3

Antibodies

Merimack Pharma: MM-121 (Schoeberl B., et al Cancer Res. 2010 Mar. 15;70(6):2485-2494)

-   -   For example, see US2011028129 SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7        and 8.        (78) RON—MST1R (Macrophage Stimulating I Receptor (c-Met-Related        Tyrosine Kinase))

Nucleotide

Genbank accession no. X70040

Genbank version no. X70040.1 GI: 36109

Genbank record update date: Feb. 2, 2011 10:17 PM

Polypeptide

Genbank accession no. CCA49634

Genbank version no. CCA49634.1 GI: 36110

Genbank record update date: Feb. 2, 2011 10:17 PM

Cross-References

Ronsin C., et al Oncogene 8 (5), 1195-1202 (1993)

Other Information

Official Symbol: MST1R

Other Aliases: CD136, CDw136, PTK8, RON

Other Designations: MSP receptor; MST1R variant RON30; MST1R variantRON62; PTK8 protein tyrosine kinase 8; RON variant E2E3; c-met-relatedtyrosine kinase; macrophage-stimulating protein receptor; p185-Ron;soluble RON variant 1; soluble RON variant 2; soluble RON variant 3;soluble RONvariant 4

(79) EPHA2 (EPH receptor A2)

Nucleotide

Genbank accession no. BC037166

Genbank version no. BC037166.2 GI: 33879863

Genbank record update date: Mar. 6, 2012 01:59 PM

Polypeptide

Genbank accession no. AAH37166

Genbank version no. AAH37166.1 GI: 22713539

Genbank record update date: Mar. 6, 2012 01:59 PM

Cross-References

Strausberg R. L., et al Proc. Natl. Acad. Sci. U.S.A. 99 (26),16899-16903 (2002)

Other Information

Official Symbol: EPHA2

Other Aliases: ARCC2, CTPA, CTPP1, ECK

Other Designations: ephrin type-A receptor 2; epithelial cell receptorprotein tyrosine kinase; soluble EPHA2 variant 1; tyrosine-proteinkinase receptor ECK

Antibodies

Medimmune: 1C1 (Lee J W., et al Clin Cancer Res. 2010 May 1;16(9):2562-2570)

-   -   For example, see US20090304721A1 FIGS. 7 and 8.

(80) CD20—MS4A1 (Membrane-Spanning 4-Domains, Subfamily A, Member 1)Nucleotide

Genbank accession no. M27394

Genbank version no. M27394.1 GI: 179307

Genbank record update date: Nov. 30, 2009 11:16 AM

Polypeptide

Genbank accession no. AAA35581

Genbank version no. AAA35581.1 GI: 179308

Genbank record update date: Nov. 30, 2009 11:16 AM

Cross-References

Tedder T. F., et al Proc. Natl. Acad. Sci. U.S.A. 85 (1), 208-212 (1988)

Other Information

Official Symbol: MS4A1

Other Aliases: B1, Bp35, CD20, CVID5, LEU-16, MS4A2, S7

Other Designations: B-lymphocyte antigen CD20; B-lymphocyte cell-surfaceantigen B1; CD20 antigen; CD20 receptor; leukocyte surface antigenLeu-16

Antibodies

Genentech/Roche: Rituximab—Abdulla N E., et al BioDrugs. 2012 Apr. 1;26(2):71-82.

-   -   For example, see U.S. Pat. No. 5,736,137, ATCC deposit No.        HB-69119.

GSK/Genmab: Ofatumumab—Nightingale G., et al Ann Pharmacother. 2011October; 45(10):1248-55.

-   -   For example, see US20090169550A1 SEQ ID NOs: 2, 4 and 5.

Immunomedics: Veltuzumab—Goldenberg D M., et al Leuk Lymphoma. 2010 May;51(5):747-55.

-   -   For example, see U.S. Pat. No. 7,919,273B2 SEQ ID NOs: 1, 2, 3,        4, 5 and 6.

(81) Tenascin C—TNC (Tenascin C) Nucleotide

Genbank accession no. NM_002160

Genbank version no. NM_002160.3 GI: 340745336

Genbank record update date: Sep. 23, 2012 02:33 PM

Polypeptide

Genbank accession no. NP_002151

Genbank version no. NP_002151.2 GI: 153946395

Genbank record update date: Sep. 23, 2012 02:33 PM

Cross-References

Nies D. E., et al J. Biol. Chem. 266 (5), 2818-2823 (1991); Siri A., etal Nucleic Acids Res. 19 (3), 525-531 (1991)

Other Information

Official Symbol: TNC

Other Aliases: 150-225, GMEM, GP, HXB, JI, TN, TN-C

Other Designations: GP 150-225; cytotactin;glioma-associated-extracellular matrix antigen; hexabrachion (tenascin);myotendinous antigen; neuronectin; tenascin; tenascin-C isoform14/AD1/16

Antibodies

Philogen: G11 (von Lukowicz T., et al J Nucl Med. 2007 April;48(4):582-7) and F16 (Pedretti M., et al Lung Cancer. 2009 April;64(1):28-33)

-   -   For example, see U.S. Pat. No. 7,968,685 SEQ ID NOs: 29, 35, 45        and 47.

(82) FAP (Fibroblast Activation Protein, Alpha) Nucleotide

Genbank accession no. U09278

Genbank version no. U09278.1 GI: 1888315

Genbank record update date: Jun. 23, 2010 09:22 AM

Polypeptide

Genbank accession no. AAB49652

Genbank version no. AAB49652.1 GI: 1888316

Genbank record update date: Jun. 23, 2010 09:22 AM

Cross-References

Scanlan, M. J., et al Proc. Natl. Acad. Sci. U.S.A. 91 (12), 5657-5661(1994)

Other Information

Official Symbol: FAP

Other Aliases: DPPIV, FAPA

Other Designations: 170 kDa melanoma membrane-bound gelatinase; integralmembrane serine protease; seprase

(83) DKK-1 (Dickkopf 1 homolog (Xenopus laevis)

Nucleotide

Genbank accession no. NM_012242

Genbank version no. NM_012242.2 GI: 61676924

Genbank record update date: Sep. 30, 2012 01:48 PM

Polypeptide

Genbank accession no. NP_036374

Genbank version no. NP_036374.1 GI: 7110719

Genbank record update date: Sep. 30, 2012 01:48 PM

Cross-References

Fedi P. et al J. Biol. Chem. 274 (27), 19465-19472 (1999)

Other Information

Official Symbol: DKK1

Other Aliases: UNQ492/PRO1008, DKK-1, SK

Other Designations: dickkopf related protein-1; dickkopf-1 like;dickkopf-like protein 1; dickkopf-related protein 1; hDkk-1

Antibodies

Novartis: BHQ880 (Fulciniti M., et al Blood. 2009 Jul. 9;114(2):371-379)

-   -   For example, see US20120052070A1 SEQ ID NOs: 100 and 108.

(84) CD52 (CD52 Molecule) Nucleotide

Genbank accession no. NM_001803

Genbank version no. NM_001803.2 GI: 68342029

Genbank record update date: Sep. 30, 2012 01:48 PM

Polypeptide

Genbank accession no. NP_001794

Genbank version no. NP_001794.2 GI: 68342030

Genbank record update date: Sep. 30, 2012 01:48 PM

Cross-References

Xia M. Q., et al Eur. J. Immunol. 21 (7), 1677-1684 (1991)

Other Information

Official Symbol: CD52

Other Aliases: CDW52

Other Designations: CAMPATH-1 antigen; CD52 antigen (CAMPATH-1 antigen);CDW52 antigen (CAMPATH-1 antigen); cambridge pathology 1 antigen;epididymal secretory protein E5; he5; human epididymis-specific protein5

Antibodies

Alemtuzumab (Campath)—Skoetz N., et al Cochrane Database Syst Rev. 2012Feb. 15; 2:CD008078.

-   -   For example, see Drugbank Acc. No. DB00087 (BIOD00109, BTD00109)

(85) CS1—SLAMF7 (SLAM Family Member 7) Nucleotide

Genbank accession no. NM_021181

Genbank version no. NM_021181.3 GI: 1993571

Genbank record update date: Jun. 29, 2012 11:24 AM

Polypeptide

Genbank accession no. NP_067004

Genbank version no. NP_067004.3 GI: 19923572

Genbank record update date: Jun. 29, 2012 11:24 AM

Cross-References

Boles K. S., et al Immunogenetics 52 (3-4), 302-307 (2001)

Other Information

Official Symbol: SLAMF7

Other Aliases: UNQ576/PRO1138, 19A, CD319, CRACC, CS1

Other Designations: 19A24 protein; CD2 subset 1; CD2-like receptoractivating cytotoxic cells; CD2-like receptor-activating cytotoxiccells; membrane protein FOAP-12; novel LY9 (lymphocyte antigen 9) likeprotein; protein 19A

Antibodies

BMS: elotuzumab/HuLuc63 (Benson D M., et al J Clin Oncol. 2012 Jun. 1;30(16):2013-2015)

-   -   For example, see US20110206701 SEQ ID NOs: 9, 10, 11, 12, 13,        14, 15 and 16.

(86) Endoglin—ENG (Endoglin) Nucleotide

Genbank accession no. AF035753

Genbank version no. AF035753.1 GI: 3452260

Genbank record update date: Mar. 10, 2010 06:36 PM

Polypeptide

Genbank accession no. AAC32802

Genbank version no. AAC32802.1 GI: 3452261

Genbank record update date: Mar. 10, 2010 06:36 PM

Cross-References

Rius C., et al Blood 92 (12), 4677-4690 (1998)

Official Symbol: ENG

Other Information

Other Aliases: RP11-228B15.2, CD105, END, HHT1, ORW, ORW1

Other Designations: CD105 antigen

(87) Annexin A1—ANXA1 (Annexin A1) Nucleotide

Genbank accession no. X05908

Genbank version no. X05908.1 GI: 34387

Genbank record update date: Feb. 2, 2011 10:02 AM

Polypeptide

Genbank accession no. CCA29338

Genbank version no. CCA29338.1 GI: 34388

Genbank record update date: Feb. 2, 2011 10:02 AM

Cross-References

Wallner B. P., et al Nature 320 (6057), 77-81 (1986)

Other Information

Official Symbol: ANXA1

Other Aliases: RP11-71A24.1, ANX1, LPC1

Other Designations: annexin I (lipocortin I); annexin-1; calpactin II;calpactin-2; chromobindin-9; lipocortin I; p35; phospholipase A2inhibitory protein

(88) V-CAM (CD106)—VCAM1 (Vascular Cell Adhesion Molecule 1) Nucleotide

Genbank accession no. M60335

Genbank version no. M60335.1 GI: 340193

Genbank record update date: Jun. 23, 2010 08:56 AM

Polypeptide

Genbank accession no. AAA61269

Genbank version no. AAA61269.1 GI: 340194

Genbank record update date: Jun. 23, 2010 08:56 AM

Cross-References

Hession C., et al J. Biol. Chem. 266 (11), 6682-6685 (1991)

Other Information

Official Symbol VCAM1

Other Aliases: CD106, INCAM-100

Other Designations: CD106 antigen; vascular cell adhesion protein 1

Antibody Sequences Anti-Integrin αvβ6 RHAB6.2 (SEQ ID NO: 5)QVQLVQSGSELKKPGASVKISCKASGFAFTDSYMHWVRQAPGQGLEWMGWIDPENGDTEYAPKFQGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCTRGTPTAVPNLRGDLQVLAQKVAGPYPFDYWGQGTLVTVSS RHCB6.2 (SEQ ID NO: 6)QVQLVQSGAEVKKPGASVKVSCKASGYTFIDSYMHWVRQAPGQRLEWMGWIDPENGDTEYAPKFQGRVTITTDTSASTAYMELSSLRSEDTAVYYCARGTPTAVPNLRGDLQVLAQKVAGPYPFDYWGQGTLVTVSS RHF (SEQ ID NO: 7)QVQLVQSGAEVKKPGASVKVSCKASGFNFIDSYMHWVRQAPGQRLEWMGWIDPENGDTEYAPKFQGRVTFTTDTSASTAYMELSSLRSEDTAVYYCNEGTPTGPYYFDYWGQGTLV TVSS RHFB6(SEQ ID NO: 8) QVQLVQSGAEVKKPGASVKVSCKASGFNFIDSYMHWVRQAPGQRLEWMGWDPENGDTEYAPKFQGRVTFTTDTSASTAYMELSSLRSEDTAVYYCNEGTPTAVPNLRGDLQVLAQKVAGPYYFDYWGQGTLVTVSS RHAY100bP (SEQ ID NO: 9)QVQLVQSGSELKKPGASVKISCKASGFAFTDSYMHWVRQAPGQGLEWMGWIDPENGDTEYAPKFQGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCTRGTPTGPYPFDYWGQGTLVTV SS RKF(SEQ ID NO: 10)ENVLTQSPGTLSLSPGERATLSCSASSSVSYMHWFQQKPGQAPRLLIYSTSNLASGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQRSSYPLTFGGGTKVEIK RKFL36L50 (SEQ ID NO: 11)ENVLTQSPGTLSLSPGERATLSCSASSSVSYMHWLQQKPGQAPRLLIYLTSNLASGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQRSSYPLTFGGGTKVEIK RKC (SEQ ID NO: 12)EIVLTQSPGTLSLSPGERATLSCSASSSVSYMHWFQQKPGQAPRLLIYSTSNLASGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQRSSYPLTFGGGTKVEIK Anti-CD33 CD33 Hum195 VH(SEQ ID NO: 13)QVQLVQSGAEVKKPGSSVKVSCKASGYTFTDYNMHWVRQAPGQGLEWIGYIYPYNGGTGYNQKFKSKATITADESTNTAYMELSSLRSEDTAVYYCARGRPAMDYWGQGTLVTVSSCD33 Hum195 VK (SEQ ID NO: 14)DIQMTQSPSSLSASVGDRVTITCRASESVDNYGISFMNWFQQKPGKAPKLLIYAASNQGSGVPSRFSGSGSGTDFTLTISSLQPDDFATYYCQQSKEVPWTFGQGTKVEIK Anti-CD19CD19 B4 resurfaced VH (SEQ ID NO: 15)QVQLVQPGAEVVKPGASVKLSCKTSGYTFTSNWMHWVKQRPGQGLEWIGEIDPSDSYTNYNQNFKGKAKLTVDKSTSTAYMEVSSLRSDDTAVYYCARGSNPYYYAMDYWGQGTSV TVSSCD19 B4 resurfaced VK (SEQ ID NO: 16)EIVLTQSPAIMSASPGERVTMTCSASSGVNYMHWYQQKPGTSPRRWIYDTSKLASGVPARFSGSGSGTSYSLTISSMEPEDAATYYCHQRGSYTFGGGTKLEIK Anti-Her2Herceptin VH chain (SEQ ID NO: 1)EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVT VSSHerceptin VL chain (SEQ ID NO: 2)DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK Anti-CD25Simulect VK (also known as Basiliximab) (SEQ ID NO: 17)QIVSTQSPAIMSASPGEKVTMTCSASSSRSYMQWYQQKPGTSPKRWIYDTSKLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCHQRSSYTFGGGTKLEIK Simulect VH(SEQ ID NO: 18)QLQQSGTVLARPGASVKMSCKASGYSFTRYWMHWIKQRPGQGLEWIGAIYPGNSDTSYNQKFEGKAKLTAVTSASTAYMELSSLTHEDSAVYYCSRDYGYYFDFWGQGTTLTVSS Anti-PSMADeimmunised VH ‘1 (SEQ ID NO: 19)EVQLVQSGPEVKKPGATVKISCKTSGYTFTEYTIHWVKQAPGKGLEWIGNINPNNGGTTYNQKFEDKATLTVDKSTDTAYMELSSLRSEDTAVYYCAAGWNFDYWGQGTLLTVSSDeimmunised VK ‘1 (SEQ ID NO: 20)DIQMTQSPSSLSTSVGDRVTLTCKASQDVGTAVDWYQQKPGPSPKLLIYWASTRHTGIPSRFSGSGSGTDFTLTISSLQPEDFADYYCQQYNSYPLTFGPGTKVDIK Deimmunised VH1 ‘5(SEQ ID NO: 21)EVKLVESGGGLVQPGGSMKLSCVASGFTFSNYWMNWVRQAPGKGLEWVAEIRSQSNNFATHYAESVKGRVTISRDDSKSIVYLQMNNLRAEDTGVYYCTRRWNNFWGQGTTVTVSSDeimmunised VH2 ‘5 (SEQ ID NO: 22)EVKLVESGGGLVQPGGSLKLSCVASGFTFSNYWMNWVRQAPGKGLEWVAEIRSQSNNFATHYAESVKGRVTISRDDSKSIVYLQMNNLRAEDTAVYYCTRRWNNFWGQGTTVTVSSDeimmunised VH3 ‘5 (SEQ ID NO: 23)EVQLVESGGGLVQPGGSLKLSCVASGFTFSNYWMNWVRQAPGKGLEWVAEIRSQSNNFATHYAESVKGRVTISRDDSKSIVYLQMNNLRAEDTAVYYCTRRWNNFWGQGTTVTVSSDeimmunised VH4 ‘5 (SEQ ID NO: 24)EVQLVESGGGLVQPGGSLKLSCVASGFTFSNYWMNWVRQAPGKGLEWVAEIRSQSNNFATHYAESVKGRFTISRDDSKSIVYLQMNNLRAEDTAVYYCTRRWNNFWGQGTTVTVSSDeimmunised VK1 ‘5 (SEQ ID NO: 25)NIVMTQFPSSMSASVGDRVTITCKASENVGTYVSWYQQKPDQSPKMLIYGASNRFTGVPDRFTGSGSATDFTLTISSLQTEDLADYYCGQSYTFPYTFGQGTKLEMK Deimmunised VK2 ‘5(SEQ ID NO: 26)NIVMTQFPSSMSASVGDRVTITCKASENVGTYVSWYQQKPDQSPKMLIYGASNRFTGVPDRFSGSGSGTDFTLTISSLQAEDLADYYCGQSYTFPYTFGQGTKLEIK Deimmunised VK3 ‘5(SEQ ID NO: 27)NIQMTQFPSAMSASVGDRVTITCKASENVGTYVSWYQQKPDQSPKMLIYGASNRFTGVPDRFSGSGSGTDFTLTISSLQAEDLADYYCGQSYTFPYTFGQGTKLEIK Deimmunised VK4 ‘5(SEQ ID NO: 28)NIQMTQFPSAMSASVGDRVTITCKASENVGTYVSWYQQKPDQSPKMLIYGASNRFTGVPDRFSGSGSGTDFTLTISSLQAEDEADYYCGQSYTFPYTFGQGTKLEIK Deimmunised VK DI ‘5(SEQ ID NO: 29)NIVMTQFPKSMSASAGERMTLTCKASENVGTYVSWYQQKPTQSPKMLIYGASNRFTGVPDRFSGSGSGTDFILTISSVQAEDLVDYYCGQSYTFPYTFGGGTKLEMK Deimmunised VH DI ‘5(SEQ ID NO: 30)EVKLEESGGGLVQPGGSMKISCVASGFTFSNYWMNWVRQSPEKGLEWVAEIRSQSNNFATHYAESVKGRVIISRDDSKSSVYLQMNSLRAEDTAVYYCTRRWNNFWGQGTTVTVSSHumanised RHA ‘5 (SEQ ID NO: 31)EVQLVESGGGLVQPGGSLKLSCAASGFTFSNYWMNWVRQASGKGLEWVGEIRSQSNNFATHYAESVKGRFTISRDDSKNTAYLQMNSLKTEDTAVYYCTRRWNNFWGQGTTVTVSSHumanised RHB ‘5 (SEQ ID NO: 32)EVKLVESGGGLVQPGGSLKLSCAASGFTFSNYWMNVVVRQASGKGLEWVAEIRSQSNNFATHYAESVKGRVIISRDDSKNTVYLQMNSLRTEDTAVYYCTRRWNNFWGQGTTVTVSSHumanised RHC ‘5 (SEQ ID NO: 33)EVQLVESGGGLVQPGGSLKLSCAASGFTFSNYWMNVWRQASGKGLEVVVAEIRSQSNNFATHYAESVKGRVIISRDDSKNTVYLQMNSLRTEDTAVYYCTRRWNNFWGQGTTVTVSSHumanised RHD ‘5 (SEQ ID NO: 34)EVKLVESGGGLVQPGGSLKLSCAASGFTFSNYWMNVWRQASGKGLEWVGEIRSQSNNFATHYAESVKGRVIISRDDSKNTVYLQMNSLRTEDTAVYYCTRRWNNFWGQGTTVTVSSHumanised RHE ‘5 (SEQ ID NO: 35)EVKLVESGGGLVQPGGSLKLSCAASGFTFSNYWMNVWRQASGKGLEWVAEIRSQSNNFATHYAESVKGRFTISRDDSKNTVYLQMNSLRTEDTAVYYCTRRWNNFWGQGTTVTVSSHumanised RHF ‘5 (SEQ ID NO: 36)EVKLVESGGGLVQPGGSLKLSCAASGFTFSNYWMNVWRQASGKGLEWVAEIRSQSNNFATHYAESVKGRVIISRDDSKNTAYLQMNSLRTEDTAVYYCTRRWNNFWGQGTTVTVSSHumanised RHG ‘5 (SEQ ID NO: 37)EVKLVESGGGLVQPGGSLKLSCAASGFTFSNYWMNVWRQASGKGLEWVAEIRSQSNNFATHYAESVKGRVIISRDDSKNTAYLQMNSLRTEDTAVYYCTRRWNNFWGQGTTVTVSSHumanised RKA ‘5 (SEQ ID NO: 38)DIQMTQSPSSVSASVGDRVTITCKASENVGTYVSWYQQKPGTAPKLLIYGASNRFTGVPSRFSGSGSATDFTLTINNLQPEDFATYYCGQSYTFPYTFGQGTKVEIK Humanised RKB ‘5(SEQ ID NO: 39)DIQMTQSPSSVSASVGDRVTITCKASENVGTYVSWYQQKPGTAPKLLIYGASNRFTGVPSRFSGSGSATDFTLTINNLQPEDFATYYCGQSYTFPYTFGQGTKVEIK Humanised RKC ‘5(SEQ ID NO: 40)DIQMTQSPSSVSASVGDRVTITCKASENVGTYVSWYQQKPGTAPKMLIYGASNRFTGVPSRFSGSGSATDFTLTINNLQPEDFATYYCGQSYTFPYTFGQGTKVEIK Humanised RKD ‘5(SEQ ID NO: 41)DIQMTQSPSSVSASVGDRVTITCKASENVGTYVSWYQQKPGTAPKMLIYGASNRFTGVPSRFSGSGSATDFTLTINNLQPEDFATYYCGQSYTFPYTFGQGTKVEIK Humanised RKE ‘5(SEQ ID NO: 42)NIVMTQSPSSVSASVGDRVTITCKASENVGTYVSWYQQKPGTAPKLLIYGASNRFTGVPDRFTGSGSATDFILTINNLQPEDFATYYCGQSYTFPYTFGQGTKVEIK Humanised RKF ‘5(SEQ ID NO: 43)NIVMTQSPSSVSASVGDRVTITCKASENVGTYVSWYQQKPGTAPKMLIYGASNRFTGVPSRFSGSGSATDFILTINNLQPEDFATYYCGQSYTFPYTFGQGTKVEIK Humanised RKG ‘5(SEQ ID NO: 44)NIVMTQSPSSVSASVGDRVTITCKASENVGTYVSWYQQKPGTAPKMLIYGASNRFTGVPDRFTGSGSATDFTLTINNLQPEDFATYYCGQSYTFPYTFGQGTKVEIK

The parent antibody may also be a fusion protein comprising analbumin-binding peptide (ABP) sequence (Dennis et al. (2002) “AlbuminBinding As A General Strategy For Improving The Pharmacokinetics OfProteins” J Biol Chem. 277:35035-35043; WO 01/45746). Antibodies of theinvention include fusion proteins with ABP sequences taught by: (i)Dennis et al (2002) J Biol Chem. 277:35035-35043 at Tables III and IV,page 35038; (ii) US 2004/0001827 at [0076]; and (iii) WO 01/45746 atpages 12-13, and all of which are incorporated herein by reference.

In one embodiment, the antibody has been raised to target specific thetumour related antigen α_(v)β₆.

The cell binding agent may be labelled, for example to aid detection orpurification of the agent either prior to incorporation as a conjugate,or as part of the conjugate. The label may be a biotin label. In anotherembodiment, the cell binding agent may be labelled with a radioisotope.

Connection of Linker Unit to Ligand Unit

The Ligand unit is connected to the Linker unit through a disulfidebond.

In one embodiment, the connection between the Ligand unit and the DrugLinker is formed between a thiol group of a cysteine residue of theLigand unit and a maleimide group of the Drug Linker unit.

The cysteine residues of the Ligand unit may be available for reactionwith the functional group of the Linker unit to form a connection. Inother embodiments, for example where the Ligand unit is an antibody, thethiol groups of the antibody may participate in interchain disulfidebonds. These interchain bonds may be converted to free thiol groups bye.g. treatment of the antibody with DTT prior to reaction with thefunctional group of the Linker unit.

In some embodiments, the cysteine residue is an introduced into theheavy or light chain of an antibody. Positions for cysteine insertion bysubstitution in antibody heavy or light chains include those describedin Published U.S. Application No. 2007-0092940 and International PatentPublication WO2008070593, which are incorporated herein.

Methods of Treatment

The compounds of the present invention may be used in a method oftherapy. Also provided is a method of treatment, comprisingadministering to a subject in need of treatment atherapeutically-effective amount of a conjugate of formula II. The term“therapeutically effective amount” is an amount sufficient to showbenefit to a patient. Such benefit may be at least amelioration of atleast one symptom. The actual amount administered, and rate andtime-course of administration, will depend on the nature and severity ofwhat is being treated. Prescription of treatment, e.g. decisions ondosage, is within the responsibility of general practitioners and othermedical doctors.

A conjugate may be administered alone or in combination with othertreatments, either simultaneously or sequentially dependent upon thecondition to be treated. Examples of treatments and therapies include,but are not limited to, chemotherapy (the administration of activeagents, including, e.g. drugs; surgery; and radiation therapy.

Pharmaceutical compositions according to the present invention, and foruse in accordance with the present invention, may comprise, in additionto the active ingredient, i.e. a conjugate of formula I, apharmaceutically acceptable excipient, carrier, buffer, stabiliser orother materials well known to those skilled in the art. Such materialsshould be non-toxic and should not interfere with the efficacy of theactive ingredient. The precise nature of the carrier or other materialwill depend on the route of administration, which may be oral, or byinjection, e.g. cutaneous, subcutaneous, or intravenous.

Pharmaceutical compositions for oral administration may be in tablet,capsule, powder or liquid form. A tablet may comprise a solid carrier oran adjuvant. Liquid pharmaceutical compositions generally comprise aliquid carrier such as water, petroleum, animal or vegetable oils,mineral oil or synthetic oil. Physiological saline solution, dextrose orother saccharide solution or glycols such as ethylene glycol, propyleneglycol or polyethylene glycol may be included. A capsule may comprise asolid carrier such a gelatin.

For intravenous, cutaneous or subcutaneous injection, or injection atthe site of affliction, the active ingredient will be in the form of aparenterally acceptable aqueous solution which is pyrogen-free and hassuitable pH, isotonicity and stability. Those of relevant skill in theart are well able to prepare suitable solutions using, for example,isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection,Lactated Ringer's Injection. Preservatives, stabilisers, buffers,antioxidants and/or other additives may be included, as required.

The Conjugates can be used to treat proliferative disease and autoimmunedisease. The term “proliferative disease” pertains to an unwanted oruncontrolled cellular proliferation of excessive or abnormal cells whichis undesired, such as, neoplastic or hyperplastic growth, whether invitro or in vivo.

Examples of proliferative conditions include, but are not limited to,benign, pre-malignant, and malignant cellular proliferation, includingbut not limited to, neoplasms and tumours (e.g., histocytoma, glioma,astrocyoma, osteoma), cancers (e.g. lung cancer, small cell lung cancer,gastrointestinal cancer, bowel cancer, colon cancer, breast carinoma,ovarian carcinoma, prostate cancer, testicular cancer, liver cancer,kidney cancer, bladder cancer, pancreatic cancer, brain cancer, sarcoma,osteosarcoma, Kaposi's sarcoma, melanoma), leukemias, psoriasis, bonediseases, fibroproliferative disorders (e.g. of connective tissues), andatherosclerosis. Other cancers of interest include, but are not limitedto, haematological; malignancies such as leukemias and lymphomas, suchas non-Hodgkin lymphoma, and subtypes such as DLBCL, marginal zone,mantle zone, and follicular, Hodgkin lymphoma, AML, and other cancers ofB or T cell origin.

Examples of autoimmune disease include the following: rheumatoidarthritis, autoimmune demyelinative diseases (e.g., multiple sclerosis,allergic encephalomyelitis), psoriatic arthritis, endocrineophthalmopathy, uveoretinitis, systemic lupus erythematosus, myastheniagravis, Graves' disease, glomerulonephritis, autoimmune hepatologicaldisorder, inflammatory bowel disease (e.g., Crohn's disease),anaphylaxis, allergic reaction, Sjögren's syndrome, type I diabetesmellitus, primary biliary cirrhosis, Wegener's granulomatosis,fibromyalgia, polymyositis, dermatomyositis, multiple endocrine failure,Schmidt's syndrome, autoimmune uveitis, Addison's disease, adrenalitis,thyroiditis, Hashimoto's thyroiditis, autoimmune thyroid disease,pernicious anemia, gastric atrophy, chronic hepatitis, lupoid hepatitis,atherosclerosis, subacute cutaneous lupus erythematosus,hypoparathyroidism, Dressler's syndrome, autoimmune thrombocytopenia,idiopathic thrombocytopenic purpura, hemolytic anemia, pemphigusvulgaris, pemphigus, dermatitis herpetiformis, alopecia arcata,pemphigoid, scleroderma, progressive systemic sclerosis, CREST syndrome(calcinosis, Raynaud's phenomenon, esophageal dysmotility,sclerodactyly, and telangiectasia), male and female autoimmuneinfertility, ankylosing spondolytis, ulcerative colitis, mixedconnective tissue disease, polyarteritis nedosa, systemic necrotizingvasculitis, atopic dermatitis, atopic rhinitis, Goodpasture's syndrome,Chagas' disease, sarcoidosis, rheumatic fever, asthma, recurrentabortion, anti-phospholipid syndrome, farmer's lung, erythemamultiforme, post cardiotomy syndrome, Cushing's syndrome, autoimmunechronic active hepatitis, bird-fancier's lung, toxic epidermalnecrolysis, Alport's syndrome, alveolitis, allergic alveolitis,fibrosing alveolitis, interstitial lung disease, erythema nodosum,pyoderma gangrenosum, transfusion reaction, Takayasu's arteritis,polymyalgia rheumatica, temporal arteritis, schistosomiasis, giant cellarteritis, ascariasis, aspergillosis, Sampter's syndrome, eczema,lymphomatoid granulomatosis, Behcet's disease, Caplan's syndrome,Kawasaki's disease, dengue, encephalomyelitis, endocarditis,endomyocardial fibrosis, endophthalmitis, erythema elevatum et diutinum,psoriasis, erythroblastosis fetalis, eosinophilic faciitis, Shulman'ssyndrome, Felty's syndrome, filariasis, cyclitis, chronic cyclitis,heterochronic cyclitis, Fuch's cyclitis, IgA nephropathy,Henoch-Schonlein purpura, graft versus host disease, transplantationrejection, cardiomyopathy, Eaton-Lambert syndrome, relapsingpolychondritis, cryoglobulinemia, Waldenstrom's macroglobulemia, Evan'ssyndrome, and autoimmune gonadal failure.

In some embodiments, the autoimmune disease is a disorder of Blymphocytes (e.g., systemic lupus erythematosus, Goodpasture's syndrome,rheumatoid arthritis, and type I diabetes), Thl-lymphocytes (e.g.,rheumatoid arthritis, multiple sclerosis, psoriasis, Sjögren's syndrome,Hashimoto's thyroiditis, Graves' disease, primary biliary cirrhosis,Wegener's granulomatosis, tuberculosis, or graft versus host disease),or Th2-lymphocytes (e.g., atopic dermatitis, systemic lupuserythematosus, atopic asthma, rhinoconjunctivitis, allergic rhinitis,Omenn's syndrome, systemic sclerosis, or chronic graft versus hostdisease). Generally, disorders involving dendritic cells involvedisorders of Thl-lymphocytes or Th2-lymphocytes. In some embodiments,the autoimmunie disorder is a T cell-mediated immunological disorder.

In some embodiments, the amount of the Conjugate administered rangesfrom about 0.01 to about 10 mg/kg per dose. In some embodiments, theamount of the Conjugate administered ranges from about 0.01 to about 5mg/kg per dose. In some embodiments, the amount of the Conjugateadministered ranges from about 0.05 to about 5 mg/kg per dose. In someembodiments, the amount of the Conjugate administered ranges from about0.1 to about 5 mg/kg per dose. In some embodiments, the amount of theConjugate administered ranges from about 0.1 to about 4 mg/kg per dose.In some embodiments, the amount of the Conjugate administered rangesfrom about 0.05 to about 3 mg/kg per dose. In some embodiments, theamount of the Conjugate administered ranges from about 0.1 to about 3mg/kg per dose. In some embodiments, the amount of the Conjugateadministered ranges from about 0.1 to about 2 mg/kg per dose.

Drug Loading

The drug loading (p) is the average number of PBD drugs per cell bindingagent, e.g. antibody. Where the compounds of the invention are bound tocysteines, drug loading may range from 1 to 8 drugs (D) per cell bindingagent, i.e. where 1, 2, 3, 4, 5, 6, 7, and 8 drug moieties arecovalently attached to the cell binding agent. Compositions ofconjugates include collections of cell binding agents, e.g. antibodies,conjugated with a range of drugs, from 1 to 8. Where the compounds ofthe invention are bound to lysines, drug loading may range from 1 to 80drugs (D) per cell binding agent, although an upper limit of 40, 20, 10or 8 may be preferred. Compositions of conjugates include collections ofcell binding agents, e.g. antibodies, conjugated with a range of drugs,from 1 to 80, 1 to 40, 1 to 20, 1 to 10 or 1 to 8.

The average number of drugs per antibody in preparations of ADC fromconjugation reactions may be characterized by conventional means such asUV, reverse phase HPLC, HIC, mass spectroscopy, ELISA assay, andelectrophoresis. The quantitative distribution of ADC in terms of p mayalso be determined. By ELISA, the averaged value of p in a particularpreparation of ADC may be determined (Hamblett et al (2004) Clin. CancerRes. 10:7063-7070; Sanderson et al (2005) Clin. Cancer Res. 11:843-852).However, the distribution of p (drug) values is not discernible by theantibody-antigen binding and detection limitation of ELISA. Also, ELISAassay for detection of antibody-drug conjugates does not determine wherethe drug moieties are attached to the antibody, such as the heavy chainor light chain fragments, or the particular amino acid residues. In someinstances, separation, purification, and characterization of homogeneousADC where p is a certain value from ADC with other drug loadings may beachieved by means such as reverse phase HPLC or electrophoresis. Suchtechniques are also applicable to other types of conjugates.

For some antibody-drug conjugates, p may be limited by the number ofattachment sites on the antibody. For example, an antibody may have onlyone or several cysteine thiol groups, or may have only one or severalsufficiently reactive thiol groups through which a linker may beattached. Higher drug loading, e.g. p>5, may cause aggregation,insolubility, toxicity, or loss of cellular permeability of certainantibody-drug conjugates.

Typically, fewer than the theoretical maximum of drug moieties areconjugated to an antibody during a conjugation reaction. An antibody maycontain, for example, many lysine residues that do not react with theDrug Linker. Only the most reactive lysine groups may react with anamine-reactive linker reagent. Also, only the most reactive cysteinethiol groups may react with a thiol-reactive linker reagent. Generally,antibodies do not contain many, if any, free and reactive cysteine thiolgroups which may be linked to a drug moiety. Most cysteine thiolresidues in the antibodies of the compounds exist as disulfide bridgesand must be reduced with a reducing agent such as dithiothreitol (DTT)or TCEP, under partial or total reducing conditions. The loading(drug/antibody ratio) of an ADC may be controlled in several differentmanners, including: (i) limiting the molar excess of Drug Linkerrelative to antibody, (ii) limiting the conjugation reaction time ortemperature, and (iii) partial or limiting reductive conditions forcysteine thiol modification.

Certain antibodies have reducible interchain disulfides, i.e. cysteinebridges. Antibodies may be made reactive for conjugation with linkerreagents by treatment with a reducing agent such as DTT(dithiothreitol). Each cysteine bridge will thus form, theoretically,two reactive thiol nucleophiles. Additional nucleophilic groups can beintroduced into antibodies through the reaction of lysines with2-iminothiolane (Traut's reagent) resulting in conversion of an amineinto a thiol. Reactive thiol groups may be introduced into the antibody(or fragment thereof) by engineering one, two, three, four, or morecysteine residues (e.g., preparing mutant antibodies comprising one ormore non-native cysteine amino acid residues). U.S. Pat. No. 7,521,541teaches engineering antibodies by introduction of reactive cysteineamino acids.

Cysteine amino acids may be engineered at reactive sites in an antibodyand which do not form intrachain or intermolecular disulfide linkages(Junutula, et al., 2008b Nature Biotech., 26(8):925-932; Dornan et al(2009) Blood 114(13):2721-2729; U.S. Pat. Nos. 7,521,541; 7,723,485;WO2009/052249). The engineered cysteine thiols may react with linkerreagents or the drug-linker reagents of the present invention which havethiol-reactive, electrophilic groups such as maleimide or alpha-haloamides to form ADC with cysteine engineered antibodies and the PBD drugmoieties. The location of the drug moiety can thus be designed,controlled, and known. The drug loading can be controlled since theengineered cysteine thiol groups typically react with thiol-reactivelinker reagents or drug-linker reagents in high yield. Engineering anIgG antibody to introduce a cysteine amino acid by substitution at asingle site on the heavy or light chain gives two new cysteines on thesymmetrical antibody. A drug loading near 2 can be achieved with nearhomogeneity of the conjugation product ADC.

Where more than one nucleophilic or electrophilic group of the antibodyreacts with a drug-linker intermediate, or linker reagent followed bydrug moiety reagent, then the resulting product is a mixture of ADCcompounds with a distribution of drug moieties attached to an antibody,e.g. 1, 2, 3, etc. Liquid chromatography methods such as polymericreverse phase (PLRP) and hydrophobic interaction (HIC) may separatecompounds in the mixture by drug loading value. Preparations of ADC witha single drug loading value (p) may be isolated, however, these singleloading value ADCs may still be heterogeneous mixtures because the drugmoieties may be attached, via the linker, at different sites on theantibody.

Thus the antibody-drug conjugate compositions of the invention includemixtures of antibody-drug conjugate compounds where the antibody has oneor more PBD drug moieties and where the drug moieties may be attached tothe antibody at various amino acid residues.

In one embodiment, the average number of dimer pyrrolobenzodiazepinegroups per cell binding agent is in the range 1 to 20. In someembodiments the range is selected from 1 to 8, 2 to 8, 2 to 6, 2 to 4,and 4 to 8.

In some embodiments, there is one dimer pyrrolobenzodiazepine group percell binding agent.

General Synthetic Routes

The synthesis of PBD compounds is extensively discussed in the followingreferences, which discussions are incorporated herein by reference:

-   a) WO 00/12508 (pages 14 to 30);-   b) WO 2005/023814 (pages 3 to 10);-   c) WO 2004/043963 (pages 28 to 29); and-   d) WO 2005/085251 (pages 30 to 39).

Synthesis Route

Compounds of the present invention of formula I where R²⁰ and R²¹ form anitrogen-carbon double bond between the nitrogen and carbon atoms towhich they are bound can be synthesised from a compound of Formula II:

where R⁶, R⁷, R⁹, R^(6′), R^(7′), R^(9′), R^(11b), Y, Y′ and R″ are asdefined for compounds of formula I, and R^(LL) is a precursor ofR^(L)—this method is particularly applicable to compounds of formula Iwhere R^(L) is of formula IIIa. For these compounds, R^(LL) willtypically be a portion of R^(L), such as a group of formula IIIa′:

In such as case, the reaction involves adding the group G^(L). Thesecond required step is removal of the Prot^(O) group.

The compounds of Formula 2 may be made by deprotecting the R^(LL) groupof compounds of Formula 3:

where R⁶, R⁷, R⁹, R^(6′), R^(7′), R^(9′), R^(11b), Y, Y′ and R″ are asdefined for compounds of formula I, R^(LL-Prot) is a protected versionof R^(LL), and the Prot^(N) represents a simple nitrogen protectinggroup (e.g. Fmoc, Boc) that is orthogonal to the R^(LL) protectinggroup.

Compounds of formula 3 may be made by ring-closure of compounds ofFormula 4:

where the ring closure is carried out by oxidation, e.g. Swern.

Compounds of formula 4 can be synthesised from compounds of formula 5:

by a step-wise addition of two protecting groups. This can be achievedby simple protection of the amino group which will result in the iminobond in the final compound (e.g. by Fmoc, Boc), followed by installationof a desired protecting group at the other amino group.

Compounds of formula I where R^(L) is of formula IIIb, may besynthesised in a similar manner, although the complete R^(L) group maybe installed starting from a compound of Formula 5, rather than with theuse of a protected precursor.

Compounds of Formula 5 can be synthesised by known methods, such asthose disclosed in WO 2011/130598.

Alternatively, compounds of Formula 4 can be synthesised by a monomericroute, as shown in example 3.

Compounds of the present invention of formula I where R²⁰ and R²¹ do notform a nitrogen-carbon double bond between the nitrogen and carbon atomsto which they are bound can by made by modifications to the aboveroutes.

Synthesis of Drug Conjugates

Conjugates can be prepared as previously described. Antibodies can beconjugated to the Drug Linker compound as described in Doronina et al.,Nature Biotechnology, 2003, 21, 778-784). Briefly, antibodies (4-5mg/mL) in PBS containing 50 mM sodium borate at pH 7.4 are reduced withtris(carboxyethyl)phosphine hydrochloride (TCEP) at 37° C. The progressof the reaction, which reduces interchain disulfides, is monitored byreaction with 5,5′-dithiobis(2-nitrobenzoic acid) and allowed to proceeduntil the desired level of thiols/mAb is achieved. The reduced antibodyis then cooled to 0° C. and alkylated with 1.5 equivalents of maleimidedrug-linker per antibody thiol. After 1 hour, the reaction is quenchedby the addition of 5 equivalents of N-acetyl cysteine. Quencheddrug-linker is removed by gel filtration over a PD-10 column. The ADC isthen sterile-filtered through a 0.22 μm syringe filter. Proteinconcentration can be determined by spectral analysis at 280 nm and 329nm, respectively, with correction for the contribution of drugabsorbance at 280 nm. Size exclusion chromatography can be used todetermine the extent of antibody aggregation, and RP-HPLC can be used todetermine the levels of remaining NAC-quenched drug-linker.

Further Preferences

The following preferences may apply to all aspects of the invention asdescribed above, or may relate to a single aspect. The preferences maybe combined together in any combination.

In some embodiments, R^(6′), R^(7′), R^(9′), and Y′ are selected fromthe same groups as R⁶, R⁷, R⁹, and Y respectively. In some of theseembodiments, R^(6′), R^(7′), R^(9′), and Y′ are the same as R⁶, R⁷, R⁹,and Y respectively.

N10′-C11′

In some embodiment, R²⁰ is H, and R²¹ is OH, OR^(A), where R^(A) is C₁₋₄alkyl. In some of these embodiments, R²¹ is OH. In others of theseembodiments, R²¹ is OR^(A), where R^(A) is C₁₋₄ alkyl. In some of theseembodiments, R^(A) is methyl.

In some embodiments, R²⁰ and R²¹ form a nitrogen-carbon double bondbetween the nitrogen and carbon atoms to which they are bound.

In some embodiments, R²⁰ is H and R²¹ is SO_(z)M, where z is 2 or 3 andM is a monovalent pharmaceutically acceptable cation. In some of theseembodiments, M is a monovalent pharmaceutically acceptable cation, andmay be Na⁺. Furthermore, in some embodiments z is 3.

In some embodiments, R²⁰ is H and R²¹ is H.

In some embodiments where R²⁰ is (d-iii), there may be an additionalnitro group on the benzene ring, e.g. ortho to R^(Z).

In some embodiments, R²¹ is OH or OR^(A), where R^(A) is C₁₋₄ alkyl andR²⁰ is selected from:

—C(═O)—X₁—NHC(═O)X₂—NH— represent a dipeptide. The amino acids in thedipeptide may be any combination of natural amino acids. The dipeptidemay be the site of action for cathepsin-mediated cleavage.

In one embodiment, the dipeptide, —C(═O)—X₁—NHC(═O)X₂—NH—, is selectedfrom:

-   -   Phe-Lys-,    -   Val-Ala-,    -   Val-Lys-,    -   Ala-Lys-,    -   Val-Cit-,    -   Phe-Cit-,    -   Leu-Cit-,    -   Ile-Cit-,    -   Phe-Arg-,    -   Trp-Cit-        where Cit is citrulline.

Preferably, the dipeptide, —C(═O)—X₁—NHC(═O)X₂—NH—, is selected from:

-   -   Phe-Lys-,    -   Val-Ala-,    -   Val-Lys-,    -   Ala-Lys-,    -   Val-Cit-.

Most preferably, the dipeptide, —C(═O)—X₁—NHC(═O)X₂—NH—, is -Phe-Lys- or-Val-Ala-.

Other dipeptide combinations may be used, including those described byDubowchik et al., Bioconjugate Chemistry, 2002, 13,855-869, which isincorporated herein by reference.

In one embodiment, the amino acid side chain is derivatised, whereappropriate. For example, an amino group or carboxy group of an aminoacid side chain may be derivatised.

In one embodiment, an amino group NH₂ of a side chain amino acid, suchas lysine, is a derivatised form selected from the group consisting ofNHR and NRR′.

In one embodiment, a carboxy group COOH of a side chain amino acid, suchas aspartic acid, is a derivatised form selected from the groupconsisting of COOR, CONH₂, CONHR and CONRR′.

In one embodiment, the amino acid side chain is chemically protected,where appropriate. The side chain protecting group may be a group asdiscussed above. The present inventors have established that protectedamino acid sequences are cleavable by enzymes. For example, it has beenestablished that a dipeptide sequence comprising a Boc sidechain-protected Lys residue is cleavable by cathepsin.

Protecting groups for the side chains of amino acids are well known inthe art and are described in the Novabiochem Catalog. Additionalprotecting group strategies are set out in Protective Groups in OrganicSynthesis, Greene and Wuts.

Possible side chain protecting groups are shown below for those aminoacids having reactive side chain functionality:

-   -   Arg: Z, Mtr, Tos;    -   Asn: Trt, Xan;    -   Asp: Bzl, t-Bu;    -   Cys: Acm, Bzl, Bzl-OMe, Bzl-Me, Trt;    -   Glu: Bzl, t-Bu;    -   Gin: Trt, Xan;    -   His: Boc, Dnp, Tos, Trt;    -   Lys: Boc, Z-Cl, Fmoc, Z, Alloc;    -   Ser: Bzl, TBDMS, TBDPS;    -   Thr: Bz;    -   Trp: Boc;    -   Tyr: Bzl, Z, Z-Br.

In one embodiment, the side chain protection is selected to beorthogonal to a group provided as, or as part of, a capping group, wherepresent. Thus, the removal of the side chain protecting group does notremove the capping group, or any protecting group functionality that ispart of the capping group.

In other embodiments of the invention, the amino acids selected arethose having no reactive side chain functionality. For example, theamino acids may be selected from: Ala, Gly, lie, Leu, Met, Phe, Pro, andVal.

It is particularly preferred in the present invention, that if L¹comprises a dipeptide, then —C(═O)—X₁—NHC(═O)X₂—NH— is the samedipeptide.

Other preferred R²⁰ groups include:

Dimer Link

In some embodiments, Y and Y′ are both O.

In some embodiments, R″ is a C₃₋₇ alkylene group with no substituents.In some of these embodiments, R″ is a C₃, C₅ or C₇ alkylene. Inparticular, R″ may be a C₃ or C₅ alkylene.

In other embodiments, R″ is a group of formula:

where r is 1 or 2.

The phenylene group may be replaced by a pyridylene group.

R⁶ to R⁹

In some embodiments, R⁹ is H.

In some embodiments, R⁶ is selected from H, OH, OR, SH, NH₂, nitro andhalo, and may be selected from H or halo. In some of these embodimentsR⁶ is H.

In some embodiments, R⁷ is selected from H, OH, OR, SH, SR, NH₂, NHR,NRR′, and halo. In some of these embodiments R⁷ is selected from H, OHand OR, where R is selected from optionally substituted C₁₋₇ alkyl,C₃₋₁₀ heterocyclyl and C₅₋₁₀ aryl groups. R may be more preferably aC₁₋₄ alkyl group, which may or may not be substituted. A substituent ofinterest is a C₅₋₆ aryl group (e.g. phenyl). Particularly preferredsubstituents at the 7-positions are OMe and OCH₂Ph. Other substituentsof particular interest are dimethylamino (i.e. —NMe₂); —(OC₂H4)_(q)OMe,where q is from 0 to 2; nitrogen-containing C₆ heterocyclyls, includingmorpholino, piperidinyl and N-methyl-piperazinyl.

These embodiments and preferences apply to R^(9′), R^(6′) and R^(7′)respectively.

R^(11b)

In some embodiments, R^(11b) is OH.

In some embodiments, R^(11b) is OR^(A), where R^(A) is C₁₋₄ alkyl. Insome of these embodiments, R^(A) is methyl.

In some embodiments of the first aspect of the present invention are offormula Ia, Ib or Ic:

where R^(1a) is selected from methyl and benzyl;R^(L) and R^(11b) are as defined above.

These embodiments and preferences also apply to the second aspect of theinvention.

Linker (R^(L))

In some embodiments, R^(L) is of formula IIIa.

In some embodiments, R^(LL) is of formula IIIa′.

G^(L)

G^(L) may be selected from

where Ar represents a C₅₋₆ arylene group, e.g. phenylene.

In some embodiments, G^(L) is selected from G^(L1-1) and G^(L1-2). Insome of these embodiments, G^(L) is G^(L1-1).

G^(LL)

G^(LL) may be selected from:

where Ar represents a C₅₋₆ arylene group, e.g. phenylene.

In some embodiments, G^(LL) is selected from G^(LL1-1) and G^(LL1-2). Insome of these embodiments, G^(LL) is G^(LL1-1).

X

X is:

where a=0 to 5, b=0 to 16, c=0 or 1, d=0 to 5.

a may be 0, 1, 2, 3, 4 or 5. In some embodiments, a is 0 to 3. In someof these embodiments, a is 0 or 1. In further embodiments, a is 0.

b may be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16. Insome embodiments, b is 0 to 12. In some of these embodiments, b is 0 to8, and may be 0, 2, 4 or 8.

c may be 0 or 1.

d may be 0, 1, 2, 3, 4 or 5. In some embodiments, d is 0 to 3. In someof these embodiments, d is 1 or 2. In further embodiments, d is 2.

In some embodiments of X, a is 0, c is 1 and d is 2, and b may be from 0to 8. In some of these embodiments, b is 0, 4 or 8.

Q

In one embodiment, Q is an amino acid residue. The amino acid may anatural amino acids or a non-natural amino acid.

In one embodiment, Q is selected from: Phe, Lys, Val, Ala, Cit, Leu,lie, Arg, and Trp, where Cit is citrulline.

In one embodiment, Q comprises a dipeptide residue. The amino acids inthe dipeptide may be any combination of natural amino acids andnon-natural amino acids. In some embodiments, the dipeptide comprisesnatural amino acids. Where the linker is a cathepsin labile linker, thedipeptide is the site of action for cathepsin-mediated cleavage. Thedipeptide then is a recognition site for cathepsin.

In one embodiment, Q is selected from:

-   -   ^(CO)-Phe-Lys-^(NH),    -   ^(CO)-Val-Ala-^(NH),    -   ^(CO)-Val-Lys-^(NH),    -   ^(CO)-Ala-Lys-^(NH),    -   ^(CO)-Val-Cit-^(NH),    -   ^(CO)-Phe-Cit-^(NH),    -   ^(CO)-Leu-Cit-^(NH),    -   ^(CO)-Ile-Cit-^(NH),    -   ^(CO)-Phe-Arg-^(NH), and    -   ^(CO)-Trp-Cit-^(NH);        where Cit is citrulline.

Preferably, Q is selected from:

-   -   ^(CO)-Phe-Lys-^(NH),    -   ^(CO)-Val-Ala-^(NH),    -   ^(CO)-Val-Lys-^(NH),    -   ^(CO)-Ala-Lys-^(NH),    -   ^(CO)-Val-Cit-^(NH).

Most preferably, Q is selected from ^(CO)-Phe-Lys-^(NH),^(CO)-Val-Cit-^(NH) and ^(CO)-Val-Ala-^(NH).

Other dipeptide combinations of interest include:

-   -   ^(CO)-Gly-Gly-^(NH)    -   ^(CO)-Pro-Pro-^(NH), and    -   ^(CO)-Val-Glu-^(NH).

Other dipeptide combinations may be used, including those described byDubowchik et al., Bioconjugate Chemistry, 2002, 13,855-869, which isincorporated herein by reference.

In some embodiments, Q^(X) is a tripeptide residue. The amino acids inthe tripeptide may be any combination of natural amino acids andnon-natural amino acids. In some embodiments, the tripeptide comprisesnatural amino acids. Where the linker is a cathepsin labile linker, thetripeptide is the site of action for cathepsin-mediated cleavage. Thetripeptide then is a recognition site for cathepsin.

In one embodiment, the amino acid side chain is chemically protected,where appropriate. The side chain protecting group may be a group asdiscussed below. Protected amino acid sequences are cleavable byenzymes. For example, a dipeptide sequence comprising a Boc sidechain-protected Lys residue is cleavable by cathepsin.

Protecting groups for the side chains of amino acids are well known inthe art and are described in the Novabiochem Catalog, and as describedabove.

In some embodiments, R^(L) is of formula IIIb.

In some embodiments, R^(LL) is formula IIIb′.

R^(L1) and R^(L2) are independently selected from H and methyl, ortogether with the carbon atom to which they are bound form acyclopropylene or cyclobutylene group.

In some embodiments, both R^(L1) and R^(L2) are H.

In some embodiments, R^(L1) is H and R^(L2) is methyl.

In some embodiments, both R^(L1) and R^(L2) are methyl.

In some embodiments, R^(L1) and R^(L2) together with the carbon atom towhich they are bound form a cyclopropylene group.

In some embodiments, R^(L1) and R^(L2) together with the carbon atom towhich they are bound form a cyclobutylene group.

In the group IIIb, in some embodiments, e is 0. In other embodiments, eis 1 and the nitro group may be in any available position of the ring.In some of these embodiments, it is in the ortho position. In others ofthese embodiments, it is in the para position.

In one particular embodiment, the first aspect of the inventioncomprises a compound of formula Id:

where Q is selected from:

(a) —CH₂—; (b) —C₃H—; and

(c)

In one particular embodiment, the second aspect of the invention, theDrug linker (D^(L)) is of formula (Id′):

where Q is selected from:

(a) —CH₂—;

(b) —C₃H₆—; and(c)

In some embodiments of the present invention, the C11 substituent may bein the following stereochemical arrangement relative to neighbouringgroups:

In other embodiments, the C11 substituent may be in the followingstereochemical arrangement relative to neighbouring groups:

EXAMPLES General Information

Flash chromatography was performed using a Biotage Isolera 1™ usinggradient elution starting from either 88% hexane/EtOAc or 99.9% DCM/MeOHuntil all UV active components (detection at 214 and 254 nm) eluted fromthe column. The gradient was manually held whenever substantial elutionof UV active material was observed. Fractions were checked for purityusing thin-layer chromatography (TLC) using Merck Kieselgel 60 F254silica gel, with fluorescent indicator on aluminium plates.Visualisation of TLC was achieved with UV light or iodine vapour unlessotherwise stated. Extraction and chromatography solvents were bought andused without further purification from VWR U.K. All fine chemicals werepurchased from Sigma-Aldrich or TCI Europe unless otherwise stated.Pegylated reagents were obtained from Quanta biodesign US via StratechUK.

¹H and ¹³C NMR spectra were obtained on a Bruker Avance® 400spectrometer. Coupling constants are quoted in hertz (Hz). Chemicalshifts are recorded in parts per million (ppm) downfield fromtetramethylsilane. Spin multiplicities are described as s (singlet), bs(broad singlet), d (doublet), t (triplet), and m (multiplet).

The analytical LC/MS conditions (for reaction monitoring and puritydetermination) were as follows: Positive mode electrospray massspectrometry was performed using a Shimadzu Nexera®/Prominence®LCMS-2020. Mobile phases used were solvent A (H₂O with 0.1% formic acid)and solvent B (CH₃CN with 0.1% formic acid). Gradient for routine3-minute run: Initial composition 5% B held over 25 seconds, thenincreased from 5% B to 100% B over a 1 minute 35 second period. Thecomposition was held for 50 seconds at 100% B, then returned to 5% B in5 seconds and held there for 5 seconds. The total duration of thegradient run was 3.0 minutes. Gradient for 15-minute run: Initialcomposition 5% B held over 1 minute, then increased from 5% B to 100% Bover a 9 minute period. The composition was held for 2 minutes at 100%B, then returned to 5% B in 10 seconds and held there for 2 minutes 50seconds. The total duration of the gradient run was 15.0 minutes. Flowrate was 0.8 mL/minute (for 3-minute run) and 0.6 mL/minute (for15-minute run). Detection was at 254 nm. Columns: Waters Acquity UPLC®BEH Shield RP18 1.7 μm 2.1×50 mm at 50° C. fitted with Waters AcquityUPLC® BEH Shield RP18 VanGuard Pre-column, 130A, 1.7 μm, 2.1 mm×5 mm(routine 3-minute run); and ACE Excel 2 C18-AR, 2μ, 3.0×100 mm fittedwith Waters Acquity UPLC® BEH Shield RP18 VanGuard Pre-column, 130A, 1.7μm, 2.1 mm×5 mm (15-minute run).

The preparative HPLC conditions were as follows: Reverse-phaseultra-fast high-performance liquid chromatography (UFLC) was carried outon a Shimazdzu Prominence® machine using a Phenomenex® Gemini NX 5μ C18column (at 50° C.) 150×21.2 mm. Eluents used were solvent A (H₂O with0.1% formic acid) and solvent B (CH₃CN with 0.1% formic acid). All UFLCexperiments were performed with gradient conditions:

Method A: Initial composition 13% B increased to 60% B over a 15 minuteperiod then increased to 100% B over 2 minutes. The composition was heldfor 1 minute at 100% B, then returned to 13% B in 0.1 minute and heldthere for 1.9 minutes. The total duration of the gradient run was 20.0minutes. Flow was 20.0 mL/minute and detection was at 254 and 280 nm.

Method B: Initial composition 13% B increased to 70% B over a 17 minuteperiod and maintained over 2 minutes, then returned to 13% B in 0.1minute and held there for 1.9 minutes. The total duration of thegradient run was 20.0 minutes. Flow was 20.0 mL/minute and detection wasat 223 nm.

Method C: Initial composition 13% B increased to 75% B over a 15 minuteperiod then increased to 100% B over 2 minutes. The composition was heldfor 1 minute at 100% B, then returned to 13% B in 0.1 minute and heldthere for 1.9 minutes. The total duration of the gradient run was 20.0minutes. Flow rate was 20.0 mL/minute and detection was at 254 and 280nm.

Example 1

(a)((Propane-1,3-diylbis(oxy))bis(5-methoxy-2-nitro-4,1-phenylene))bis(((S)-2-(hydroxymethyl)pyrrolidin-1-yl)methanone)(I2)

DMF (12 drops) was added to a stirred suspension of the bis-nitrobenzoicacid 11 (10 g, 21.5 mmol) and oxalyl chloride (5.6 mL, 8.2 g, 64.5 mmol)in anhydrous DCM (150 mL).

Following initial effervescence the reaction suspension became asolution and the mixture was allowed to stir at room temperature for 16hours. The majority of solvent was removed by evaporation in vacuo andthe resulting concentrated solution was re-dissolved in a minimum amountof dry DCM and triturated with diethyl ether. The resulting yellowprecipitate was collected by vacuum filtration, washed with cold diethylether and dried for 1 hour in a vacuum oven at 40° C. The solid acidchloride was added portion-wise to a stirred suspension of(S)-(+)-2-pyrrolidinemethanol (5.0 g, 4.9 mL, 49.5 mmol) and TEA (15.0mL, 10.9 g, 108 mmol) in DCM (100 mL) at −40° C. (dry ice/CH₃CN). After1 hour stirring, the reaction was complete as judged by LC/MS withexclusively desired product at retention time 1.33 minutes, ES+ m/z 655[M+Na]⁺, 633 [M+H]⁺. The mixture was diluted with DCM (100 mL) andwashed with 1N HCl (2×50 mL), saturated NaHCO₃ (3×40 mL), brine (50 mL),dried (MgSO₄), filtered and the solvent evaporated in vacuo to give thepure product I2 as a yellow solid (13.6 g, 100% yield).

(b)((2S,2′S)-(4,4′-(propane-1,3-diylbis(oxy))bis(5-methoxy-2-nitrobenzoyl))bis(pyrrolidine-1,2-diyl))bis(methylene)diacetate (I3)

A solution of Ac₂O (4.47 mL, 4.83 g, 47.3 mmol) in dry DCM (25 mL) wasadded drop-wise to a stirred solution of the bis-alcohol 12 (13.6 g,21.5 mmol), DMAP (263 mg, 2.15 mmol) and pyridine (4.17 mL, 4.08 g, 51.6mmol) in dry DCM (125 mL) at 0° C. (ice/acetone) under an argonatmosphere. The reaction mixture was allowed to warm-up and after 1 hourat room temperature analysis by LC/MS revealed completion of reactionand clean conversion to desired product at retention time 1.55 minutes,ES+ m/z 740 [M+Na]⁺, 717 [M+H]⁺. The mixture was diluted with DCM (20mL) and washed with 1N HCl (2×100 mL), H₂O (25 mL), brine (50 mL), dried(MgSO₄), filtered and the solvent evaporated in vacuo to give the crudebis-acetate 13 as a yellow solid (14.4 g, 94% yield) which was ofsatisfactory purity to be carried through to the next step withoutfurther purification.

(c)((2S,2′S)-(4,4′-(propane-1,3-diylbis(oxy))bis(2-amino-5-methoxybenzoyl))bis(pyrrolidine-1,2-diyl))bis(methylene)diacetate (I4)

A sample of 10% Pd—C (132 mg) was treated carefully with EtOAc (10 mL)to give a slurry which was added to a solution of the nitro compound I3(1.32 g, 1.84 mmol) in EtOAc (20 mL) and EtOH (30 mL) in a hydrogenationvessel. Using Parr® apparatus, the mixture was treated with hydrogen gasto 10 psi and shaken at room temperature then degassed in vacuo, thisprocess was repeated a further two times. The vessel was filled withhydrogen gas to 45 psi, shaken and the pressure maintained uponconsumption of hydrogen. Analysis by LC/MS showed the reaction wasincomplete after 3 hours and was left shaking at 45 psi for 3 days (theweekend) after which time satisfactory conversion to product wasachieved, retention time=1.32 minutes, ES+ m/z 657 [M+H]⁺. The reactionmixture was degassed in vacuo and then filtered through a Celite® pad.The filtrate was evaporated in vacuo, the resulting residue re-dissolvedin DCM (30 mL), dried (MgSO₄), filtered and the solvent evaporated invacuo to give the crude bis-aniline I4 as a yellowish foam (1.1 g, 91%yield) which contained an 8% impurity but was carried through to thenext step without further purification.

(d)((S)-1-(4-(3-(4-((S)-2-(acetoxymethyl)pyrrolidine-1-carbonyl)-5-((tert-butoxycarbonyl)amino)-2-methoxyphenoxy)propoxy)-2-amino-5-methoxybenzoyl)pyrrolidin-2-yl)methylacetate (I5)

Boc₂O (330 mg, 1.51 mmol) was added to a stirred solution of thebis-aniline 14 (1.1 g, 1.68 mmol) in dry THF (10 mL). The reactionmixture was heated and stirred at 75° C. for 16 hours. Analysis by LC/MSrevealed desired mono Boc product I5 at retention time 1.58 minutes, I%=50, ES+ m/z 779 [M+Na]⁺, 757 [M+H]⁺ along with unreacted startingmaterial at retention time 1.32 minutes, I %=30, and bis-Boc material atretention time 1.81 minutes, I %=21, ES+ m/z 879 [M+Na]⁺, 857 [M+H]⁺.The reaction mixture was allowed to cool to room temperature and the THFremoved by evaporation in vacuo. Purification by Isolera™ (DCM/MeOH,SNAP Ultra 50 g, 100 mL per minute) provided the mono Boc product I5 asan orange foam (519 mg, 46% yield based on Boc₂O, eluting at 97%DCM/MeOH) unreacted bis-aniline 14 (285 mg, eluting at 95% DCM/MeOH) andbis-Boc (248 mg, eluting at 98% DCM/MeOH).

(e)((S)-1-(4-(3-(4-((S)-2-(acetoxymethyl)pyrrolidine-1-carbonyl)-5-((((4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3-methylbutanamido)propanamido)benzyl)oxy)carbonyl)amino)-2-methoxyphenoxy)propoxy)-2-((tert-butoxycarbonyl)amino)-5-methoxybenzoyl)pyrrolidin-2-yl)methylacetate (I7)

Triphosgene (380 mg, 1.28 mmol) was added to a stirred solution of themono Boc product 15 (2.69 g, 3.56 mmol) and TEA (1.09 mL, 791 mg, 7.83mmol) in dry DCM (30 mL) at room temperature. After stirring for 10minutes under argon, analysis by LC/MS revealed complete conversion toisocyanate (sampled in MeOH to give methyl carbamate, retention time1.66 minutes, ES+ m/z 837 [M+Na]⁺, 815 [M+H]⁺). The mixture was treatedwith additional TEA (740 μL, 539 mg, 5.34 mmol) followed by the additionof linker 16 (1.34 g, 3.56 mmol). After 2 hours stirring under argon,LC/MS revealed satisfactory conversion to carbamate 17 (retention time1.74 minutes, (ES+) m/z 1182 [M+Na]⁺, 1160 [M+H]⁺). The mixture wasdiluted with DCM (80 mL) and washed with saturated NH₄Cl (2×30 mL), H₂O(30 mL), brine (50 mL), dried (MgSO₄), filtered and evaporated in vacuoto give the crude product. Purification by Isolera™ (Hexane/EtOAc, SNAPUltra 100 g, 100 mL per minute) provided the pure carbamate 17 (elutingat 65% Hexane/EtOAc) as a yellow foam (2.95 g, 71% yield).

(f) tert-butyl(5-(3-(5-((((4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3-methylbutanamido)propanamido)benzyl)oxy)carbonyl)amino)-4-((S)-2-(hydroxymethyl)pyrrolidine-1-carbonyl)-2-methoxyphenoxy)propoxy)-2-((S)-2-(hydroxymethyl)pyrrolidine-1-carbonyl)-4-methoxyphenyl)carbamate(I8)

Solid K₂CO₃ (1.75 g, 12.7 mmol) was added to a stirred solution of theacetate-protected compound I7 (2.93 g, 2.53 mmol) in MeOH (60 mL) andH₂O (12 mL). After 1 hour stirring at room temperature the reaction wasdeemed to be complete as judged by LC/MS with desired product atretention time 1.57 minutes, ES+ m/z 1098 [M+Na]⁺, 1076 [M+H]⁺. The MeOHwas removed by evaporation in vacuo and the resulting residue waspartitioned between water (75 mL) and DCM (75 mL). The layers wereseparated and the aqueous phase was extracted with DCM (3×25 mL). Thecombined organic layers were washed with water (3×50 mL), brine (60 mL),dried (MgSO₄), filtered and evaporated in vacuo to provide the crudeproduct. Purification by Isolera™ (DCM/MeOH, SNAP Ultra 100 g, 100 mLper minute) the bis-alcohol 18 (eluting at 97% DCM/MeOH) as a white foam(2.44 g, 90% yield).

(g)4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3-methylbutanamido)propanamido)benzyl(11S,11aS)-8-(3-(((11S,11aS)-10-(tert-butoxycarbonyl)-11-hydroxy-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-11-hydroxy-7-methoxy-5-oxo-2,3,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-10(5H)-carboxylate(I9)

A solution of anhydrous DMSO (710 μL, 780 mg, 9.99 mmol) in dry DCM (20mL) was added drop-wise to a stirred solution of oxalyl chloride (2.72mL of a 2.0M solution in DCM, 5.44 mmol) in dry DCM (20 mL) at −45° C.(dry ice/CH₃CN) under an argon atmosphere. After 15 minutes stirring at−45° C., the reaction mixture was treated drop-wise with a solution ofthe bis-alcohol 18 (2.44 g, 2.27 mmol) in dry DCM (30 mL). Afterstirring at −45° C. for a further 1 hour, the reaction mixture wastreated drop-wise with a solution of TEA (3.16 mL, 2.29 g, 22.7 mmol) indry DCM (20 mL). The reaction mixture was allowed to warm to roomtemperature over a period of 1.5 hours and diluted with DCM (100 mL)then washed with saturated NH₄Cl (2×50 mL), saturated NaHCO₃ (50 mL),water (30 mL), brine (50 mL), dried (MgSO₄), filtered and evaporated invacuo to give the crude product. Purification by Isolera™ (DCM/MeOH,SNAP Ultra 100 g, 100 mL per minute) gave the cyclised compound I9(eluting at 95.7% DCM/MeOH) as a yellowish foam (1.61 g, 66% yield):LC/MS I9 at retention time 1.46 minutes, ES+ m/z 1072 [M+H]⁺, 1094[M+Na]⁺.

(h)4-((S)-2-((S)-2-amino-3-methylbutanamido)propanamido)benzyl(11S,11aS)-8-(3-(((11S,11aS)-10-(tert-butoxycarbonyl)-11-hydroxy-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-11-hydroxy-7-methoxy-5-oxo-2,3,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-10(5H)-carboxylate(I10)

Pd(PPh₃)₄ (6.47 mg, 5.6 μmol) was added to a stirred solution ofpyrrolidine (29 μL, 25 mg, 0.35 mmol) and the Alloc compound I9 (300 mg,0.28 mmol) in dry DCM (10 mL). After stirring for 4 hours under argon atroom temperature, analysis by LC/MS revealed reaction completion withdesired product observed at retention time 1.10 minutes, ES+, m/z 1010[M+Na]⁺, 988 [M+H]⁺. The reaction mixture was diluted with DCM (30 mL)then washed with saturated NH₄Cl (2×20 mL), brine (30 mL), dried(MgSO₄), filtered and evaporated in vacuo to give the crude product.Trituration with diethyl ether followed by evaporation in vacuo gave thecrude amine 110 (261 mg, 95% yield) which was carried through to thenext step without further purification or analysis.

(i)tert-butyl(11S,11aS)-8-(3-(((11S,11aS)-10-(((4-((2S,5S)-37-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-5-isopropyl-2-methyl-4,7,35-trioxo-10,13,16,19,22,25,28,31-octaoxa-3,6,34-triazaheptatriacontanamido)benzyl)oxy)carbonyl)-1-hydroxy-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-11-hydroxy-7-methoxy-5-oxo-2,3,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-10(5H)-carboxylate(111)

EDCl (56 mg, 0.29 mmol) was added to a stirred solution ofMAL-dPEG®₈-acid (172 mg, 0.29 mmol, Stratech Scientific Limited) and theamine 110 (261 mg, 0.26 mmol) in dry DCM (10 mL) at room temperature.The reaction mixture was stirred under an argon atmosphere for 2.5 hoursat which point analysis by LC/MS showed complete conversion to desiredproduct at retention time 1.38 minutes, ES+ m/z 1585 [M+Na]⁺, 1563[M+H]⁺. The reaction mixture was diluted with DCM (30 mL) and washedwith H₂O (20 mL), brine (2×20 mL), dried (MgSO₄), filtered andevaporated in vacuo to provide the crude product. Purification byIsolera™ (DCM/MeOH, SNAP Ultra 25 g, 75 mL per minute) gave the amideI11 (eluting at 91% DCM/MeOH) as a white foam (277 mg, 67% yield).

(j)4-((2S,5S)-37-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-5-isopropyl-2-methyl-4,7,35-trioxo-10,13,16,19,22,25,28,31-octaoxa-3,6,34-triazaheptatriacontanamido)benzyl(11S,11aS)-11-hydroxy-7-methoxy-8-(3-(((S)-7-methoxy-5-oxo-2,3,5,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-5-oxo-2,3,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-10(5H)-carboxylate(1)

A solution of 95:5 v/v TFA/H₂O (2 mL) was added to a crude sample of theBoc-protected compound I11 (262 mg, 0.17 mmol) at 0° C. (ice/acetone).After stirring at 0° C. for 3 hours the reaction was deemed complete asjudged by LC/MS, desired product peak at retention time 1.30 minutes,ES+ m/z 1445 [M+H]⁺. The reaction mixture was kept cold and addeddrop-wise to a chilled saturated aqueous solution of NaHCO₃ (100 mL).The mixture was extracted with DCM (3×30 mL) and the combined organiclayers washed with brine (30 mL), dried (MgSO₄), filtered and evaporatedin vacuo to provide the crude product. Purification by Isolera™(CHCl₃/MeOH, SNAP Ultra 25 g, 25 mL per minute) gave 1 (eluting at 89.6%CHCl₃/MeOH) as a yellow foam (170 mg, 70% yield). Further purificationby preparative HPLC (Method A) gave 1 as a light yellow foam (105 mg,43% yield): LC/MS (15-minute run), retention time 5.25 minutes, ES+ m/z1445 [M+H]⁺; ¹H NMR (400 MHz, d6-DMSO) δ 9.92 (s, 1H), 8.16 (d, 1H,J=6.8 Hz), 7.99 (t, 1H, J=5.7 Hz), 7.86 (d, 1H, J=8.6 Hz), 7.80 (d, 1H,J=4.5 Hz), 7.64-7.50 (m, 2H), 7.34 (s, 1H), 7.24-7.13 (m, 2H), 7.06 (s,1H), 7.00 (s, 2H), 6.88 (s, 1H), 6.75 (s, 1H), 6.53-6.41 (m, 1H),5.52-5.41 (m, 1H), 5.13 (d, 1H, J=12.2 Hz), 4.93-4.77 (m, 1H), 4.42-4.34(m, 1H), 4.30-3.90 (m, 6H), 3.80-3.60 (m, 4H), 3.80 (s, 3H), 3.79 (s,3H), 3.60 (t, 4H, J=7.3 Hz), 3.53-3.46 (m, 28H), 3.41-3.33 (m, 1H),3.32-3.29 (m, 2H, obscured by H₂O), 3.19-3.12 (m, 2H), 2.48-1.60, m,15H), 1.35-1.20 (m, 3H), 0.87 (d, 3H, J=6.6 Hz), 0.83 (d, 3H, J=6.8 Hz).

Example 2

(a)((pentane-1,5-diylbis(oxy))bis(5-methoxy-2-nitro-4,1-phenylene))bis(((S)-2-(hydroxymethyl)pyrrolidin-1-yl)methanone)(I13)

DMF (5 drops) was added to a stirred suspension of the bis-nitrobenzoicacid 112 (4.05 g, 8.192 mmol, 1.0 eq.) and oxalyl chloride (12.3 mL of2M solution, 24.57 mmol, 3.0 eq.) in anhydrous CH₂Cl₂ (65 mL). Followinginitial effervescence the reaction suspension became a solution and themixture was allowed to stir at room temperature for 16 hours. Thereaction mixture was concentrated in vacuo and the resulting solid wastriturated with Et₂O and dried in a vacuum oven at 40° C. for 3 hours.The solid acid chloride was added portion-wise to a stirred suspensionof (S)-(+)-2-pyrrolidinemethanol (1.78 mL, 18.02 mmol, 2.2 eq.) andi-Pr₂NEt (7.13 mL, 40.96 mmol, 5.0 eq.) in CH₂Cl₂ (65 mL) at −40° C.(dry ice/CH₃CN). After 1 hour stirring, the reaction temperature hadreached OC, and was complete as judged by LC/MS with exclusively desiredproduct at retention time 1.44 minutes, ES+ m/z 661 [M+H]⁺, 683 [M+Na]⁺.The mixture was diluted with CH₂Cl₂ (100 mL) and washed consecutivelywith H₂O, 1N NaOH and 1M HCl (50 mL), dried (MgSO₄), filtered and thesolvent evaporated in vacuo to give the pure product I13 as a yellowfoam (4.44 g, 82% yield), which was used without further purification.

(b)((pentane-1,5-diylbis(oxy))bis(5-methoxy-2-nitro-4,1-phenylene))bis(((S)-2-(((tert-butyldimethylsilyl)oxy)methyl)pyrrolidin-1-yl)methanone)(114)

Imidazole (2.74 g, 40.32 mmol, 6.0 eq.) then TBSCl (3.04 g, 20.16 mmol,3.0 eq.) were added portion-wise to a stirred solution of bis-alcoholI13 (4.44 g, 6.720 mmol, 1.0 eq.) under an argon atmosphere. After 90minutes, the reaction mixture was filtered and the filtrated washed withH₂O, dried over MgSO₄ and concentrated in vacuo. Flash columnchromatography (50-80% EtOAc in hexane) afforded the product I14 as ayellow foam (4.84 g, 5.443 mmol, 81% yield). LC/MS retention time=2.18min, ES+ m/z 889 [M+H]⁺, 911 [M+Na]⁺.

(c)((pentane-1,5-diylbis(oxy))bis(2-amino-5-methoxy-4,1-phenylene))bis(((S)-2-(((tert-butyldimethylsilyl)oxy)methyl)pyrrolidin-1-yl)methanone)(I15)

Zn powder was added to a stirring solution of bis-nitro compound I14(1.32 g, 1.84 mmol) in MeOH (40 mL) at 0° C. 5% HCO₂H in MeOH was addeddropwise at 0° C. and the mixture stirred for 2 hours. The reactionmixture was diluted with EtOAc and washed with sat. NaHCO₃ solution, theorganic phase dried over MgSO₄ and concentrated in vacuo. Flash columnchromatography (0-2% MeOH in CHCl₃) afforded the product I15 as a paleyellow foam (3.098 mmol, 3.736 mmol, 69% yield). LC/MS retentiontime=2.09 min, ES+ m/z 415 [M+2H]²⁺, 829 [M+H]⁺, 851 [M+Na]⁺.

(d) tert-butyl(5-((5-(5-amino-4-((S)-2-(((tert-butyldimethylsilyl)oxy)methyl)pyrrolidine-1-carbonyl)-2-methoxyphenoxy)pentyl)oxy)-2-((S)-2-(((tert-butyldimethylsilyl)oxy)methyl)pyrrolidine-1-carbonyl)-4-methoxyphenyl)carbamate(116)

Boc₂O (734 mg, 3.362 mmol) was added to a stirred solution of thebis-aniline 115 (3.098 g, 3.736 mmol) in dry THF (20 mL). The reactionmixture was stirred for 16 hours and concentrated in vacuo. Flash columnchromatography (30-50% EtOAc in hexane) provided the mono Boc productI16 as a yellow foam (1.474 g, 47% yield based on Boc₂O) unreactedbis-aniline I15 (1.043 g, 30% yield) and bis-Boc (419 mg, 15% yield,LC/MS retention time=2.37 min). LC/MS retention time of 116=2.25 min,ES⁺ m/z 929 [M+H]⁺, 951 [M+Na]⁺.

(e) tert-butyl(5-((5-(5-((((4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3-methylbutanamido)propanamido)benzyl)oxy)carbonyl)amino)-4-((S)-2-(((tert-butyldimethylsilyl)oxy)methyl)pyrrolidine-1-carbonyl)-2-hydroxyphenoxy)pentyl)oxy)-2-((S)-2-(((tert-butyldimethylsilyl)oxy)methyl)pyrrolidine-1-carbonyl)-4-methoxyphenyl)carbamate (I17)

Triphosgene (169 mg, 0.5710 mmol, 0.36 eq.) was added to a stirredsolution of the mono Boc product I16 (1.474 g, 1.586 mmol, 1.0 eq.) andEt₃N (486 μL, 3.489 mmol, 2.2 eq.) in dry CH₂Cl₂ (9 mL) at −10° C. Afterstirring for 10 minutes under argon, analysis by LC/MS revealed completeconversion to isocyanate (sampled in MeOH to give methyl carbamate,retention time 2.30 minutes, ES+ m/z 1009 [M+Na]⁺, 987 [M+H]⁺). Asolution of 16 (898 mg, 2.379 mmol, 1.5 eq.) and Et₃N (332 μL, 2.379mmol, 1.5 eq.) in dry CH₂Cl₂ (14 mL) was added. The reaction wasgradually warmed to rt and stirred for 16 h. 15 minute LC/MS analysisrevealed starting material had been consumed. The reaction mixture wasfiltered through a SiO₂ pad (5% MeOH in CH₂Cl₂ elution) to remove excess16. Flash column chromatography (20-80% EtOAc in hexane) provided 117 asa yellow foam (1.439 g, 68% yield). LC/MS retention time=2.26 min (3minute run) and 10.43 min (15 minute run) ES⁺ m/z 1355 [M+Na]⁺, 1333[M+H]⁺. A negligible amount of urea dimer was observed (LC/MS retentiontime=12.11 min, ES⁺ m/z 1906 [M+Na]⁺) which was removed in subsequentpurification steps.

(f) tert-butyl(5-((5-(5-((((4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3-methylbutanamido)propanamido)benzyl)oxy)carbonyl)amino)-2-hydroxy-4-((S)-2-(hydroxymethyl)pyrrolidine-1-carbonyl)phenoxy)pentyl)oxy)-2-((S)-2-(hydroxymethyl)pyrrolidine-1-carbonyl)-4-methoxyphenyl)carbamate (I18)

Acetic acid (124 μL, 2.160 mmol, 2.0 eq.) was added to a 1M solution ofTBAF (3.2 mL, 3.200 mmol, 3.0 eq.) and subsequently added to a stirringsolution of 117 in THF (67 mL) at 0° C. The reaction mixture was warmedto room temperature and stirred for 16 hours. LC/MS indicated reactionincomplete. TBAF (1.00 mL of 1M solution, 1 mmol, 1.0 eq.) was added andthe reaction mixture stirred for a further 24 hours. The reactionmixture was concentrated in vacuo and purified by Isolera™ (0-5% MeOH inCH₂Cl₂) to afford the product I18 as a pale yellow foam (916 mg, 77%yield). LC/MS retention time=1.62 min, ES⁺ m/z 1126 [M+Na]⁺, 1104[M+H]⁺.

(g)4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3-methylbutanamido)propanamido)benzyl(11S,11aS)-8-((5-(((11S,11aS)-10-(tert-butoxycarbonyl)-11-hydroxy-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)pentyl)oxy)-11-hydroxy-7-methoxy-5-oxo-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate(I19)

IBX (1.14 g, 1.825 mmol, 2.2 eq.) was added to a stirring solution ofdiol I18 (916 mg, 0.8296 mmol, 1.0 eq.) in DMSO. The reaction mixturewas warmed to 35° C. and stirred for 60 hours. H₂O was added and theaqueous was extracted with CHCl₃ several times. The organic extractswere combined, washed with sat. NaHCO₃ and dried over MgSO₄.Purification by Isolera™ (1-8% MeOH in CH₂Cl₂) provided 119 as an orangefoam (908 mg, 99% yield): LC/MS retention time=1.50 minutes, ES+ m/z1122 [M+Na]⁺, 1100 [M+H]⁺.

(h) 4-((S)-2-((S)-2-amino-3-methylbutanamido)propanamido)benzyl(11S,11aS)-8-((5-(((11S,11aS)-10-(tert-butoxycarbonyl)-11-hydroxy-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)pentyl)oxy)-11-hydroxy-7-methoxy-5-oxo-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate(I20)

Pd(PPh₃)₄ (15.8 mg, 13.64 μmol, 0.050 eq.) was added to a stirringsolution of pyrrolidine (56 μL, 0.6818 mmol, 2.5 eq.) and 119 (300 mg,0.2727 mmol) in CH₂Cl₂ (10 mL) under argon. After 30 minutes, sat. NH₄Clsolution was added and the mixture vigorously stirred and transferred toan Isolute® Phase Separator. The collected organic phase wasconcentrated in vacuo to afford an orange foam 120 which was usedwithout further purification.

(i) tert-butyl(11S,11aS)-8-((5-(((11S,11aS)-10-(((4-((2S,5S)-37-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-5-isopropyl-2-methyl-4,7,35-trioxo-10,13,16,19,22,25,28,31-octaoxa-3,6,34-triazaheptatriacontanamido)benzyl)oxy)carbonyl)-11-hydroxy-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)pentyl)oxy)-11-hydroxy-7-methoxy-5-oxo-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate(I21)

EDCl.HCl (117 mg, 0.29 mmol) was added to a stirred solution ofMAL-dPEG®₈-acid (360 mg, 0.6079 mmol, Stratech Scientific Limited) andamine 120 (608 mg, 0.5526 mmol) in CH₂Cl₂ (15 mL) at room temperature.The reaction mixture was stirred under an argon atmosphere for 24 hours,at which point analysis by LC/MS showed complete consumption of I20. Thereaction mixture was diluted with CH₂Cl₂ and washed successively withsat. NH₄Cl and sat. NaHCO₃, dried over MgSO₄, and concentrated in vacuoto provide the crude product. Purification by Isolera™ (4-16% MeOH inCH₂Cl₂) gave amide 121 as a white solid (77 mg, 79% purity (UVintegration @ 223 nm) 8.8% crude yield; 107 mg, 88% purity, 12% crudeyield; 224 mg, 86% purity, 25% crude yield).

(j)4-((2S,5S)-37-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-5-isopropyl-2-methyl-4,7,35-trioxo-10,13,16,19,22,25,28,31-octaoxa-3,6,34-triazaheptatriacontanamido)benzyl(11S,11aS)-11-hydroxy-7-methoxy-8-(3-(((S)-7-methoxy-5-oxo-2,3,5,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-5-oxo-2,3,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-10(5H)-carboxylate(2)

An ice-cold solution of 95:5 v/v TFA/H₂O (3 mL) was added to a crudesample of the Boc-protected compound I21 (107 mg, 67.51 μmol) at 0° C.(ice/brine). After stirring at 0° C. for 30 min, the reaction was deemedcomplete as judged by LC/MS, desired product peak at retention time 1.38minutes, ES+ m/z 737 [M+2H]²⁺, 748 [M+H+Na]²+; 1472 [M+H]⁺. The reactionmixture was kept cold and added drop-wise to a chilled saturated aqueoussolution of NaHCO₃. The mixture was extracted with CH₂Cl₂, then 10% MeOHin CH₂Cl₂, the combined organic layers dried over MgSO₄ and concentratedin vacuo to provide the crude product. This process was repeated for theother batches of 121, the crude products combined and purified bypreparative HPLC (Method B) to afford 2 as a white solid afterlyophilisation (126 mg, 33% yield, 96% purity by UV @ 223 nm): LC/MS (30minute run), retention time=10.96 minutes, ES+ m/z 1472 [M+H]⁺.

Example 3

(a) I23

This step may be carried out as in the literature (see for exampleWO2005085259A2; or Wells, et al., Bioorganic & Medicinal ChemistryLetters, 18 (2008) 2147-2151). The method involves a Parr Hydrogenationat room temperature with 10% Pd/C in EtOH. The yield is quantitative.Ethanol is removed by two evaporations (EtOAc, followed by DCM).

(b) Tert-butyl(11S,11aS)-8-((3-(bromomethyl)benzyl)oxy)-7-methoxy-5-oxo-11-((tetrahydro-2H-pyran-2-yl)oxy)-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate(I25)

A mixture of phenol 123 (4 g, 8.91 mmol, 1 eq),1,3-bis(bromomethyl)benzene 124 (9.42 g, 35.7 mmol, 4 eq), potassiumcarbonate (1.23 g, 8.91 mmol, 1 eq), and acetone (40 mL) were heated at60° C. for 5 hours. After completion was observed by LC/MS, the solidswere removed by filtration and the filtrate was concentrated to drynessunder vacuum. The residue was purified by chromatography (BiotageIsolera, 100 g Ultra, gradient EtOAc/Hexane 30/70 up to 80/20 in 12CV).Yield 4.25 g (75%). LC/MS, 3 min method, 1.82 min (ES+) m/z (relativeintensity) 631.15 ([M+H]⁺, 100), split peak: THP diastereoisomers. ¹HNMR (400 MHz, DMSO-d₆) δ 7.67-7.27 (m, 4H), 7.20-6.57 (m, 2H), 5.72-5.57(m, 1H), 5.24-4.84 (m, 3H), 4.72 (s, 2H), 3.91-3.73 (m, 4H), 3.61-3.33(m, 4H), 2.20-1.75 (m, 4H), 1.74-1.57 (m, 2H), 1.55-1.01 (m, 13H).

I28 is known in the literature (see WO2013053872A1, Compound 2, page 60)

(c) (S)-(2-(hydroxymethyl)pyrrolidin-1-yl)(5-methoxy-2-nitro-4-((triisopropylsilyl)oxy)phenyl)methanone (I29)

EDCl (12.4 g, 65 mmol, 1.2 eq) was added to a solution of acid 128 (20g, 54.1 mmol, 1 eq), and hydroxybenzotriazole hydrate (8.05 g, 59.5mmol, 1.1 eq) in dichloromethane (200 mL) at 0° C. The cold bath wasremoved and the reaction was allowed to proceed for 30 mins at roomtemperature, at which time a solution of (S)-pyrrolidin-2-ylmethanol(5.87 mL, 59.5 mmol, 1.1 eq) and triethylamine (11.32 mL, 81.1 mmol, 1.5eq) in dichloromethane (100 mL) was added rapidly at −10° C. underargon. The reaction mixture was allowed to stir at room temperature for40 min to 1 h and monitored by LC/MS and TLC (EtOAc). The solids wereremoved by filtration over celite and the organic phase was washed withcold aqueous 0.1 M HCl until the pH was measured at 4 or 5. The organicphase was then washed with water, followed by saturated aqueous sodiumbicarbonate and brine. The organic layer was dried over magnesiumsulphate, filtered and excess solvent removed by rotary evaporationunder reduced pressure. The residue was subjected to column flashchromatography (Isolera Biotage, 340 g Ultra; gradient 25/75 ethylacetate/hexane to 100/0 ethyl acetate/hexane in 6 CV). Excess solventwas removed by rotary evaporation under reduced pressure afforded thepure product I29 as a pale yellow foam (15.7 g, 64%). LC/MS 1.92 min(ES+) m/z (relative intensity) 453.15 ([M+H]⁺, 30%; 328.15, 100%); ¹HNMR (400 MHz, Chloroform-d) δ 7.70 (s, 1H), 6.77 (s, 1H), 4.57-4.24 (m,2H), 4.01-3.69 (m, 5H), 3.25-3.06 (m, 2H), 2.18 (dt, J=7.5, 5.6 Hz, 1H),1.96-1.62 (m, 3H), 1.42-1.18 (m, 3H), 1.10 (d, J=7.4 Hz, 18H).

(d) (S)-(2-(((tert-butyldimethylsilyl)oxy)methyl)pyrrolidin-1-yl)(5-methoxy-2-nitro-4-((triisopropylsilyl)oxy)phenyl)methanone (130)

t-Butyldimethylsilyl chloride (10.39 g, 68.9 mmol, 2 eq) was added to asolution of alcohol I29 (15.6 g, 34.5 mmol, 1 eq), and imidazole (5.87g, 86.2 mmol, 2.5 eq) in DCM (100 mL). The reaction mixture stirredovernight at room temperature. The reaction mixture was sequentiallywashed with water (300 mL), 0.5 M citric acid (200 mL), brine (100 mL),and dried (MgSO4). Filtration and removal of excess solvent furnishedthe crude product, which was subjected to flash column chromatography(Biotage Isolera, KP-Sil 340 g; 10/90 v/v ethyl acetate/hexane up to30/70 v/v ethyl acetate/hexane) to isolate the silyl ether 130 as athick yellow oil. Yield: 18.9 g, 97%. LC/MS 2.32 min (ES+) m/z (relativeintensity) 567.55 ([M+H]⁺, 100%)

(e) (S)-(2-amino-5-methoxy-4-((triisopropylsilyl)oxy)phenyl)(2-(((tert-butyldimethylsilyl)oxy)methyl)pyrrolidin-1-yl)methanone (I31)

A solution of nitro compound I30 (18.9 g, 33.3 mmol, 1 eq) in ethylacetate (200 mL) over 10% Pd/C (10% w/w, 1.89 g) was hydrogenated underpressure (45 psi) on a Parr apparatus for 6 h. The reaction mixture wasfiltered through celite to remove the Pd/C, and the filter pad wasrinsed with ethyl acetate. Excess solvent was removed by rotaryevaporation under reduced pressure, followed by drying under high vacuumto give amine I31 as a thick oil. LC/MS, 3 min method, 2.28 min (ES+)m/z (relative intensity) 537.30 ([M+H]⁺, 100); ¹H NMR (400 MHz,Chloroform-d) δ 6.73 (s, 1H), 6.24 (s, 1H), 4.54-4.13 (m, 3H), 4.07-3.80(m, 1H), 3.79-3.61 (m, 4H), 3.50 (dd, J=9.2, 4.2 Hz, 2H), 2.10-1.97 (m,2H), 1.92 (dt, J=11.7, 6.2 Hz, 1H), 1.80-1.65 (m, 1H), 1.24 (ddt,J=13.7, 9.9, 6.4 Hz, 3H), 1.09 (d, J=7.3 Hz, 18H), 0.90 (s, 9H), 0.04(d, J=2.8 Hz, 6H).

(f) Allyl((S)-1-(((S)-1-((4-((((2-((S)-2-(((tert-butyldimethylsilyl)oxy)methyl)pyrrolidine-1-carbonyl)-4-methoxy-5-((triisopropylsilyl)oxy)phenyl)carbamoyl)oxy)methyl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate (I32)

Triethylamine (10.1 mL, 72.4 mmol, 2.2 eq) was added to a stirredsolution of the amine I31 (17.68 g, 32.9 mmol, 1 eq) and triphosgene(3.51 g, 11.8 mmol, 0.36 eq) in dry tetrahydrofuran (180 mL) at 5° C.(ice bath). The progress of the isocyanate reaction was monitored byperiodically removing aliquots from the reaction mixture and quenchingwith methanol and performing LC/MS analysis. Once the isocyanateformation was complete a suspension of the alloc-Val-Ala-PABOH 16 (18.6g, 49.4 mmol, 1.5 eq) and triethylamine (6.88 mL, 49.4 mmol, 1.5 eq) indry tetrahydrofuran (70 mL) was rapidly added to the freshly preparedisocyanate. The reaction mixture was allowed to stir at 40° C. for 4hours. The solids were removed by filtration. Excess solvent was removedby rotary evaporation under reduced pressure. The resulting residue wasdry loaded on silica gel and subjected to manual flash columnchromatography; 40/60 v/v ethyl acetate/hexane up to 70/30 v/v ethylacetate/hexane. Pure fractions were collected and combined and excesseluent was removed by rotary evaporation under reduced pressure to givethe product I32 8.17 g (26.4%). LC/MS, 3 min method, 2.29 min (ES+) m/z(relative intensity) 962.45 ([M+Na]⁺, 100; 940.40 ([M+H]⁺, 30); ¹H NMR(400 MHz, Chloroform-d) δ 8.95 (s, 1H), 8.53 (s, 1H), 7.78 (s, 1H), 7.53(d, J=8.1 Hz, 2H), 7.32 (d, J=8.3 Hz, 2H), 6.80 (s, 1H), 6.71 (d, J=7.5Hz, 1H), 5.89 (tt, J=10.8, 5.3 Hz, 1H), 5.44-5.15 (m, 3H), 5.10 (s, 2H),4.66 (p, J=7.2 Hz, 1H), 4.62-4.53 (m, 2H), 4.32 (s, 1H), 4.08-3.86 (m,2H), 3.74 (s, 4H), 3.52 (dd, J=27.4, 7.6 Hz, 2H), 2.15 (h, J=6.8 Hz,1H), 2.09-1.85 (m, 3H), 1.71 (s, 1H), 1.46 (d, J=7.0 Hz, 3H), 1.29 (dq,J=15.0, 7.4 Hz, 3H), 1.11 (d, J=7.4 Hz, 18H), 0.95 (dd, J=14.1, 6.8 Hz,6H), 0.89 (s, 9H), 0.02 (d, J=13.1 Hz, 6H).

(g) Allyl((S)-1-(((S)-1-((4-((((2-((S)-2-(((tert-butyldimethylsilyl)oxy)methyl)pyrrolidine-1-carbonyl)-5-hydroxy-4-methoxyphenyl)carbamoyl)oxy)methyl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate (I33)

Lithium acetate (50 mg, 0.49 mmol) was added to a solution of compoundI32 (7 g, 7.44 mmol, 1 eq) in wet dimethylformamide (61.2 mL, 50:1DMF/water). After 4 hours, the reaction was complete. Excess DMF wasremoved under vacuum and the residue was diluted with ethyl acetate (300mL) and washed with 0.5M aqueous citric acid (100 mL), water (300 mL)and brine (100 mL). The organic layer was dried over magnesium sulphatefiltered and excess ethyl acetate was removed by rotary evaporationunder reduced pressure. The resulting residue was subjected to columnflash chromatography (Biotage Isolera 100 g Ultra; gradient, 40/60 to80/20 v/v ethyl acetate/hexane in 8 CV). Pure fractions were collectedand combined and excess eluent was removed by rotary evaporation underreduced pressure to give the product I33 (5.13 g, 88%). LC/MS, 3 minmethod, 1.82 min (ES+) m/z (relative intensity) 784.40 ([M+H]⁺, 100). ¹HNMR (400 MHz, Chloroform-d) δ 9.06 (s, 1H), 8.62 (s, 1H), 7.78 (s, 1H),7.45 (d, J=8.2 Hz, 2H), 7.35-7.18 (m, 2H), 6.92 (d, J=7.5 Hz, 1H), 6.80(s, 1H), 6.50 (s, 1H), 5.89 (ddd, J=16.2, 10.7, 5.4 Hz, 1H), 5.44 (d,J=8.1 Hz, 1H), 5.37-5.15 (m, 2H), 5.14-5.01 (m, 2H), 4.67 (p, J=7.1 Hz,1H), 4.63-4.50 (m, 2H), 4.33 (s, 1H), 4.13-3.89 (m, 2H), 3.81 (s, 3H),3.74-3.33 (m, 3H), 2.24-1.84 (m, 4H), 1.69 (d, J=21.2 Hz, 1H), 1.43 (d,J=7.0 Hz, 3H), 1.07-0.71 (m, 15H), 0.23-−0.20 (m, 6H).

(h) Tert-butyl(11S,11aS)-8-((3-((5-((((4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3-methylbutanamido)propanamido)benzyl)oxy)carbonyl)amino)-4-((S)-2-(((tert-butyldimethylsilyl)oxy)methyl)pyrrolidine-1-carbonyl)-2-methoxyphenoxy)methyl)benzyl)oxy)-7-methoxy-5-oxo-11-((tetrahydro-2H-pyran-2-yl)oxy)-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate(I34)

Potassium carbonate (582 mg, 4.21 mmol, 1.1 eq) was added to a solutionof 125 (2.66 g, 4.21 mmol, 1.1 eq) and phenol 133 (3 g, 3.82 mmol, 1 eq)in acetone (18 mL). The reaction was stirred for 4 hours at 63° C. Thesolids were removed by filtration over cotton wool. Acetone was removedby rotary evaporation under reduced pressure. The resulting residue wassubjected to flash column chromatography (Biotage isolera, 100 g Ultra,silica gel; gradient, 50/50 to 100/0 v/v ethyl acetate/hexane in 8 CV,elution from 83%). Pure fractions were collected and combined and excesseluent was removed by rotary evaporation under reduced pressure to givethe product I34 (4.71 g, 92%). LC/MS, 3 min method, 2.08 min (ES+) m/z(relative intensity) 1335.15 ([M+H]⁺, 50). ¹H NMR (400 MHz, DMSO-d₆) δ9.98 (s, 1H), 9.20 (s, 1H), 8.13 (d, J=7.0 Hz, 1H), 7.68-7.50 (m, 3H),7.50-7.37 (m, 3H), 7.32 (d, J=8.2 Hz, 2H), 7.28-7.01 (m, 2H), 6.86 (s,2H), 5.90 (ddd, J=16.0, 10.7, 5.2 Hz, 1H), 5.64 (t, J=9.8 Hz, 1H), 5.30(d, J=17.2 Hz, 1H), 5.23-4.84 (m, 8H), 4.57-4.36 (m, 3H), 4.11 (s, 1H),3.95-3.59 (m, 9H), 3.56-3.34 (m, 4H), 1.94 (d, J=34.0 Hz, 10H),1.74-1.06 (m, 21H), 1.01-0.59 (m, 15H), 0.03 (s, 6H).

(i) Tert-butyl(11S,11aS)-8-((3-((5-((((4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3-methylbutanamido)propanamido)benzyl)oxy)carbonyl)amino)-4-((S)-2-(hydroxymethyl)pyrrolidine-1-carbonyl)-2-methoxyphenoxy)methyl)benzyl)oxy)-7-methoxy-5-oxo-11-((tetrahydro-2H-pyran-2-yl)oxy)-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate (I35)

Tetra-n-butylammonium fluoride (1M, 6.94 mL, 6.94 mmol, 2 eq) was addedto a solution of 134 (4.63 g, 3.47 mmol, 1 eq) in tetrahydrofuran (28mL). The starting material was totally consumed after 1 h. The reactionmixture was diluted with ethyl acetate (30 mL) and washed sequentiallywith water and brine. The organic phase was dried over magnesiumsulphate filtered and excess ethyl acetate removed by rotary evaporationunder reduced pressure. The resulting residue was subjected to flashcolumn chromatography (Biotage isolera, 50 g Ultra; gradient, 98/2 to90/10 v/v ethyl acetate/methanol in 4 CV, elution from 10% methanol).Pure fractions were collected and combined and excess eluent was removedby rotary evaporation under reduced pressure to give the product I35(4.23 g, quantitative). LC/MS, 3 min, 1.75 min (ES+) m/z (relativeintensity) 1220.30 ([M+H]⁺, 100). ¹H NMR (400 MHz, DMSO-d₆) δ 9.98 (s,1H), 9.17 (s, 1H), 8.13 (d, J=7.0 Hz, 1H), 7.70-7.49 (m, 3H), 7.51-7.27(m, 6H), 7.21 (d, J=8.8 Hz, 1H), 7.15-6.58 (m, 3H), 5.90 (dt, J=10.9,5.5 Hz, 1H), 5.66 (d, J=9.3 Hz, 1H), 5.38-4.82 (m, 9H), 4.73 (t, J=5.8Hz, 1H), 4.59-4.34 (m, 3H), 4.05 (dd, J=15.4, 8.3 Hz, 1H), 3.96-3.68 (m,8H), 3.66-3.32 (m, 6H), 2.16-1.72 (m, 8H), 1.63 (d, J=9.8 Hz, 3H),1.54-1.02 (m, 18H), 0.86 (dd, J=18.2, 6.7 Hz, 6H).

(j)4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3-methylbutanamido)propanamido)benzyl(11S,11aS)-8-((3-((((11S,11aS)-10-(tert-butoxycarbonyl)-7-methoxy-5-oxo-11-((tetrahydro-2H-pyran-2-yl)oxy)-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)methyl)benzyl)oxy)-11-hydroxy-7-methoxy-5-oxo-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate(I36)

Stabilised IBX 45% (2.72 g, 4.36 mmol, 1.2 eq) was added to a solutionof 135 (4.44 g, 3.64 mmol, 1 eq) in DMSO (2.6 mL). The reaction mixturewas allowed to stir overnight. Another 0.2 eq of IBX (450 mg, 0.73 mmol,0.2 eq) was added and the solution allowed to stir for another 18 huntil reaction completion was observed by LC/MS. The solution wasprecipitated in water (250 mL) and filtered. The product was dissolvedin DCM and the residual white solid removed by filtration. The organicphase was washed with aqueous NaHCO₃, water, brine, and dried overmagnesium sulphate. The dichloromethane was removed by rotaryevaporation under reduced pressure. The resulting residue was subjectedto column flash chromatography (Biotage Isolera 100 g Ultra; gradient,99/1 to 92/8 v/v DCM/methanol in 10 CV). Pure fractions were collectedand combined and removal of excess eluent by rotary evaporation underreduced pressure afforded the product I36 (3.04 g, 69%). LC/MS, 15 minmethod Ace Excel 2, 7.89 and 7.97 min (THP diastereoisomers) (ES+) m/z(relative intensity) 1218.30 ([M]⁺, 100). ¹H NMR (400 MHz, DMSO-d₆) δ9.93 (s, 1H), 8.11 (d, J=6.9 Hz, 1H), 7.68-7.27 (m, 6H), 7.27-7.01 (m,4H), 7.01-6.32 (m, 3H), 6.02-5.81 (m, 1H), 5.71-5.57 (m, 1H), 5.57-5.40(m, 1H), 5.29 (d, J=17.2 Hz, 1H), 5.21-4.78 (m, 8H), 4.58-4.32 (m, 3H),3.99-3.68 (m, 8H), 3.58-3.31 (m, 8H), 2.23-1.72 (m, 9H), 1.72-1.04 (m,18H), 0.85 (dd, J=18.0, 6.7 Hz, 6H).

(k) 4-((S)-2-((S)-2-amino-3-methylbutanamido)propanamido)benzyl(11S,11aS)-8-((3-((((11S,11aS)-10-(tert-butoxycarbonyl)-7-methoxy-5-oxo-11-((tetrahydro-2H-pyran-2-yl)oxy)-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)methyl)benzyl)oxy)-11-hydroxy-7-methoxy-5-oxo-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate(137)

Tetrakis(triphenylphosphine)palladium(0) (11 mg, 0.01 mmol, 0.02 eq) wasadded to a solution of 136 (600 mg, 0.49 mmol, 1 eq) and pyrrolidine (51μL, 0.62 mmol, 1.25 eq) in dry dichloromethane (10 mL). The reaction wasflushed with argon three times and stirred 20 minutes at roomtemperature. Then the reaction was diluted with dichloromethane (50 mL)and washed sequentially with saturated aqueous ammonium chloride (50 mL)and brine (30 mL). The organic phase was dried over magnesium sulphatefiltered and excess dichloromethane removed by rotary evaporation underreduced pressure. The resulting residue 137 was used as a crude mixturefor the next reaction. LC/MS, 3 min method, 1.29 min (ES+) m/z (relativeintensity) 1134.35 ([M+H]⁺, 80).

(l) tert-butyl(11S,11aS)-8-((3-((((11S,11aS)-10-(((4-((2S,5S)-37-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-5-isopropyl-2-methyl-4,7,35-trioxo-10,13,16,19,22,25,28,31-octaoxa-3,6,34-triazaheptatriacontanamido)benzyl)oxy)carbonyl)-11-hydroxy-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)methyl)benzyl)oxy)-7-methoxy-5-oxo-11-((tetrahydro-2H-pyran-2-yl)oxy)-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate(I38)

1-ethyl-3-(3′-dimethylaminopropyl)carbodiimide (94 mg, 0.79 mmol, 1 eq)was added to a solution of crude 137 (558 mg, 0.49 mmol, 1 eq) andMal-(PEG)₈-acid (292 mg, 0.49 mmol, 1 eq) in chloroform (12 mL). Thereaction was degassed three times with Argon and stirred for 2 hours andthe presence of starting material was no longer observed by LC/MS. Thereaction was diluted with dichloromethane and washed sequentially withwater and brine. The organic phase was dried over magnesium sulphatefiltered and excess dichloromethane removed by rotary evaporation underreduced pressure. The resulting residue was subjected to flash columnchromatography (Biotage Isolera 50 g Ultra; 98/2 to 90/10 v/vDCM/methanol in 10 CV). Pure fractions were collected and combined andexcess eluent was removed by rotary evaporation under reduced pressureto give 138 (485 mg, 58%). LC/MS, 3 min method, 1.58 min (ES+) m/z(relative intensity) 1709.30 ([M+H]⁺, 100). ¹H NMR (400 MHz, DMSO-d₆) δ9.88 (s, 1H), 8.13 (d, J=7.0 Hz, 1H), 8.06-7.92 (m, 1H), 7.85 (d, J=8.6Hz, 1H), 7.68-7.04 (m, 9H), 6.99 (s, 2H), 6.89 (d, J=15.0 Hz, 2H), 6.52(s, 1H), 5.66 (d, J=9.4 Hz, 1H), 5.47 (d, J=8.0 Hz, 1H), 5.26-4.75 (m,6H), 4.49-4.31 (m, 1H), 4.20 (t, J=7.6 Hz, 1H), 3.80 (d, J=11.9 Hz, 6H),3.59 (t, J=7.2 Hz, 4H), 3.55-3.41 (m, 32H), 3.41-3.30 (m, 11H),3.21-3.09 (m, 3H), 2.48-2.28 (m, 4H), 2.18-1.08 (m, 24H), 0.84 (dd,J=15.0, 6.7 Hz, 5H).

(m)4-((2S,5S)-37-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-5-isopropyl-2-methyl-4,7,35-trioxo-10,13,16,19,22,25,28,31-octaoxa-3,6,34-triazaheptatriacontanamido)benzyl(11S,11aS)-11-hydroxy-7-methoxy-8-((3-((((S)-7-methoxy-5-oxo-2,3,5,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)methyl)benzyl)oxy)-5-oxo-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate(3)

A cold mixture of TFA/water (6 mL) was added to 138 (460 mg, 0.27 mmol,1 eq) and the resulting solution was allowed to stir at 0° C. for 2hours. The reaction was neutralised with saturated aqueous NaHCO₃ (200mL) and dichloromethane (50 mL). The DCM layer was washed sequentiallywith water and brine. The organic phase was dried over magnesiumsulphate filtered and excess dichloromethane removed by rotaryevaporation under reduced pressure. The resulting residue was subjectedto flash column chromatography (Biotage Isolera 50 g Ultra; 98/2 to88/12 v/v DCM/methanol in 10 CV). The pure fractions were collected,combined (154 mg, 38%), and further purified by reverse phasepreparative HPLC (Method C) (gradient up to 75/25 acetonitrile/water,0.02% formic) to give pure 3 (78 mg, 19%). LC/MS, 15 min method,Ace-Excel2, 6.18 min (ES+) m/z (relative intensity) 1506.70 ([M+H]⁺,100). ¹H NMR (400 MHz, DMSO-d₆) δ 10.07-9.79 (m, 1H), 8.14 (d, J=7.2 Hz,1H), 7.98 (t, J=5.6 Hz, 1H), 7.85 (d, J=8.6 Hz, 1H), 7.78 (d, J=4.4 Hz,1H), 7.67-7.30 (m, 7H), 7.28-7.05 (m, 3H), 6.99 (s, 2H), 6.98-6.85 (m,2H), 6.58-6.47 (m, 1H), 5.59-5.34 (m, 1H), 5.32-4.77 (m, 6H), 4.48-4.30(m, 1H), 4.28-4.08 (m, 1H), 3.88-3.75 (m, 5H), 3.75-3.55 (m, 6H),3.55-3.42 (m, 28H), 3.42-3.32 (m, 6H), 3.14 (q, J=5.8 Hz, 2H), 2.48-2.16(m, 6H), 2.08-1.77 (m, 6H), 1.36-1.17 (m, 4H), 0.84 (dd, J=15.4, 6.7 Hz,6H).

Example 4

(a)((Propane-1,3-diylbis(oxy))bis(5-methoxy-2-nitro-4,1-phenylene))bis(((S)-2-(hydroxymethyl)pyrrolidin-1-yl)methanone)(I2)

DMF (12 drops) was added to a stirred suspension of 11 (10 g, 21.5 mmol)and oxalyl chloride (5.6 mL, 8.2 g, 64.5 mmol) in anhydrous DCM (150mL). Following initial effervescence the reaction suspension became asolution and the mixture was allowed to stir at room temperature for 16hr. Majority of solvent was removed by evaporation under reducedpressure. The resulting concentrated solution was re-dissolved in aminimum amount of dry DCM and triturated with diethyl ether. The yellowprecipitate was collected by vacuum filtration, washed with cold diethylether and dried for 1 hr in a vacuum oven at 40° C. The acid chloridewas added, in portions, to a stirred suspension of(S)-(+)-2-pyrrolidinemethanol (5.0 g, 4.9 mL, 49.5 mmol) and TEA (15.0mL, 10.9 g, 108 mmol) in anhydrous DCM (100 mL) at −40° C. (dryice/CH₃CN). The resulting solution was stirred for a further 60 mins,diluted with DCM (100 mL) and washed with 1N HCl (2×50 mL), saturatedNaHCO₃ (3×40 mL), brine (50 mL), dried (MgSO₄) and the solventevaporated under vacuum to give the pure product I2 as a yellow solid(13.6 g, 100% yield). LC/MS (method A): retention time 1.33 mins (ES+)m/z 655 [M+Na]⁺, 633 [M+H]⁺ (see appendix). ¹H NMR (400 MHz, DMSO-d₆) δ1.68-1.80 (m, 2H), 1.80-2.00 (m, 6H), 2.27 (d, 2H), 3.05-3.25 (m, 4H),3.37-3.48 (m, 2H), 3.56-3.76 (m, 2H), 3.92 (s, 6H), 4.09 (dd, 2H),4.25-4.31 (m, 4H), 4.82 (t, 2H), 7.08 (s, 2H), 7.73 (s, 2H).

(b)((propane-1,3-diylbis(oxy))bis(5-methoxy-2-nitro-4,1-phenylene))bis(((S)-2-(((tert-butyldimethylsilyl)oxy)methyl)pyrrolidin-1-yl)methanone)(139)

TBS-Cl (8.12 g, 53.90 mmol) was added to a solution of 12 (15.5 g, 24.50mmol) and imidazole (4.17 g, 61.25 mmol) in DCM (300 mL) at roomtemperature under nitrogen. The resulting mixture was stirred at roomtemperature for 12 hr. Water (200 mL) was added, the organic layerremoved, and the aqueous phase extracted with DCM (2×300 mL). Thecombined organic phases were dried (Na₂SO₄) and evaporated under vacuumto afford dark residue which was purified by column chromatography (0 to2% methanol/DCM). Pure fractions were evaporated under vacuum to afford139 as a brown solid (17.0 g, 81% yield). LC/MS (method A): retentiontime 1.83 mins (ES+) m/z 861 [M+H]⁺. ¹H NMR (400 MHz, DMSO-d₆) δ 0.09(s, 12H), 0.91 (s, 18H), 1.70-1.81 (m, 2H), 1.87-1.99 (m, 6H), 2.22-2.30(m, 2H), 3.10 (t, 4H), 3.40-3.51 (m, 2H), 3.59-3.67 (m, 2H), 3.88-3.95(m, 2H), 3.91 (s, 6H), 4.28 (t, 6H), 6.96 (s, 2H), 7.72 (s, 2H).

(c) ((propane-1,3-diylbis(oxy))bis(2-amino-5-methoxy-4,I-phenylene))bis(((S)-2-(((tert-butyldimethylsilyl)oxy)methyl)pyrrolidin-1-yl)methanone)(140)

Zinc (25.8 g, 394.8 mmol) and saturated NH₄Cl (150 mL) were added to asolution of 139 (17 g, 19.74 mmol) in EtOH (300 mL) at room temperature.The resulting mixture was stirred at 50° C. for 3 hr, cooled andfiltered through a bed of celite which was then washed with EtOAc (300mL) and water (300 mL). The organic layer was removed and the aqueouslayer extracted with EtOAc (3×400 mL). The combined organic phases weredried (MgSO₄) and evaporated under vacuum to afford a yellow residuewhich was purified by column chromatography (0 to 5% methanol/DCM). Purefractions were evaporated to dryness to give 140 as a yellow solid(13.00 g, 82% yield). LC/MS (method A): retention time 2.30 mins (ES+)m/z 802.2 [M+H]⁺ 1H NMR (400 MHz, DMSO-d₆) δ 0.06 (s, 12H), 0.85 (s,18H), 1.52-1.78 (m, 2H), 1.81-2.00 (m, 6H), 2.14-2.22 (m, 2H), 3.41 (d,4H), 3.61-3.75 (m, 4H), 3.63 (s, 6H), 4.01-4.16 (m, 6H), 4.98-5.22 (m,4H), 6.40 (s, 2H), 6.66 (s, 2H).

(d) allyl(5-(3-(5-amino-4-((S)-2-(((t-butyldimethylsilyl)oxy)methyl)pyrrolidine-1-carbonyl)-2-methoxyphenoxy)propoxy)-2-((S)-2-(((t-butyldimethylsilyl)oxy)methyl)pyrrolidine-1-carbonyl)-4-methoxyphenyl)carbamate (I41)

Allyl chloroformate (784 μL, 0.9 g, 7.36 mmol) was added dropwise to asolution of 140 (5.9 g, 7.36 mmol) and pyridine (715 μL, 0.7 g, 8.84mmol) in DCM (100 mL) at 00° C. The reaction mixture was warmed to roomtemperature and stirred for a further 2 hr. The reaction mixture waswashed with 0.5M HCl (50 mL), saturated sodium hydrogen carbonate (50mL) and brine (50 mL). The solvent was removed under reduced pressureand the resulting oil purified by column chromatography; (initialelution with 50% ethyl acetate/heptane removed the bis-alloc protectedamine, this was followed by elution with ethyl acetate to remove thedesired mono-alloc protected product (141). Finally, any unreactedstarting material was removed with 5% methanol/DCM). Pure fractions wereevaporated under reduced pressure to leave 141 as a yellow solid (3.5 g,54% yield). LC/MS (method B): retention time 2.41 mins (ES+) m/z 886.5[M+H]⁺

(e) allyl(5-(3-(5-((((4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3-methylbutanamido)propanamido)benzyl)oxy)carbonyl)amino)-4-((S)-2-(((tert-butyldimethylsilyl)oxy)methyl)pyrrolidine-1-carbonyl)-2-methoxyphenoxy)propoxy)-2-((S)-2-(((tert-butyldimethylsilyl)oxy)methyl)pyrrolidine-1-carbonyl)-4-methoxyphenyl)carbamate(I42)

Triphosgene (0.41 g, 1.4 mmol) was added to a stirred solution of I41(3.5 g, 3.95 mmol) in dry THF (70 mL) at room temperature under Argon.Triethylamine (1.2 mL, 0.87 g, 8.6 mmol) was added, and the resultingmixture stirred for 10 mins. Analysis by LC/MS revealed completeconversion to isocyanate (sampled in MeOH to give methyl carbamate,retention time 2.48 mins, (ES+) m/z 944.4 [M+H]⁺). A mixture of 16 (1.64g, 4.35 mmol) and triethylamine (0.83 mL, 0.6 g, 5.9 mmol) in dry THF(30 mL) was added. The reaction mixture was stirred under argon for 2 hrat 40° C. The solvent was removed under vacuum and the residue purifiedby column chromatography (0.5 to 2.5% methanol/DCM) to leave 142 as awhite solid (3.58 g, 70% yield). LC/MS (method B): retention time 2.45mins, (ES+) m/z 1290.0 [M+H]⁺.

(f) allyl(5-(3-(5-((((4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3-methylbutanamido)propanamido)benzyl)oxy)carbonyl)amino)-4-((S)-2-(hydroxymethyl)pyrrolidine-1-carbonyl)-2-methoxyphenoxy)propoxy)-2-((S)-2-(hydroxymethyl)pyrrolidine-1-carbonyl)-4-methoxyphenyl)carbamate(I43)

1M tetrabutylammonium fluoride (6.1 mL, 6.1 mmol) was added to asolution of 142 (3.58 g, 2.78 mmol) in THF (35 mL) at room temperature.The resulting solution was stirred for 60 mins, then evaporated todryness under reduced pressure. The residue was purified by columnchromatography (2 to 5% methanol/DCM) to leave 143 as a white foam (2.95g, 98% yield). LC/MS (method B): retention time 1.70 mins, (ES+) m/z1061.3 [M+H]⁺

(g) allyl(11S,11aS)-8-(3-(((11S,11aS)-10-(((4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3-methylbutanamido)propanamido)benzyl)oxy)carbonyl)-11-hydroxy-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)propoxy)-11-hydroxy-7-methoxy-5-oxo-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate(I44)

Stahl aerobic oxidation TEMPO solution 0.2M in MeCN (5.47 mL, 1.1 mmol)followed by tetrakisacetonitrile copper (I) triflate (0.41 g, 1.1 mmol)was added to a solution of 143 (2.9 g, 2.74 mmol) in DCM (30 mL) andacetonitrile (6 mL) and stirred at 35° C. for 36 hr under an atmosphereof air. The reaction mixture was washed with water (25 mL), dried(biotage phase separator) and evaporated to dryness under reducedpressure. The residue was purified by column chromatography (3 to 6%methanol/DCM) to leave the oxidised product I44 as a white solid (2.46g, 85% yield). LC/MS (method B): retention time 1.60 mins, (ES+) m/z1057.1 [M+H]⁺.

(h) 4-((S)-2-((S)-2-amino-3-methylbutanamido)propanamido)benzyl(11S,11aS)-11-hydroxy-7-methoxy-8-(3-(((S)-7-methoxy-5-oxo-2,3,5,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)propoxy)-5-oxo-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate(I45)

Pd(Ph₃P)₄ (10 mg, 5 mol %) was added to a solution of 144 (200 mg, 0.19mmol) and pyrrolidine (40 μL, 0.34 g, 0.48 mmol) in DCM (10 mL) at roomtemperature. The resulting solution was stirred for 30 mins. Thereaction mixture was washed with saturated ammonium chloride (10 mL),dried (biotage phase separator) and evaporated to dryness under reducedpressure. The residue was then placed on a high vacuum line for 4 hr toremove traces of pyrrolidine. The resulting off-white solid was used inthe next step without further purification (160 mg, 97% yield). LC/MS(method B): retention time 1.17 mins, (ES+) m/z 871.1 [M+H]⁺.

(i)4-((2S,5S)-37-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-5-isopropyl-2-methyl-4,7,35-trioxo-10,13,16,19,22,25,28,31-octaoxa-3,6,34-triazaheptatriacontanamido)benzyl(11S,11aS)-11-hydroxy-7-methoxy-8-(3-(((S)-7-methoxy-5-oxo-2,3,5,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)propoxy)-5-oxo-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate(1)

EDCl.HCl (46 mg, 0.24 mmol) was added to a solution of 145 andMal-PEG₈-acid (130 mg, 0.22 mmol) in CHCl₃ (10 mL) and stirred at roomtemperature for 2 hr. LC/MS shows 78% starting material present. Afurther 2 eq EDCl.HCl was added in portions to push the reaction tocompletion. The reaction mixture was washed with water (10 mL), dried(Biotage PS) and evaporated to dryness, under reduced pressure, to leavea yellow solid which was purified by prep HPLC to leave the product 1 asan off-white solid (90 mg, 34% yield). LC/MS (method B): retention time1.47 mins, (ES+) m/z 1445.9 [M+H]⁺.

Example 5 (i)4-((29S,32S)-1-azido-29-isopropyl-32-methyl-27,30-dioxo-3,6,9,12,15,18,21,24-octaoxa-28,31-diazatritriacontan-33-amido)benzyl(11S,11aS)-11-hydroxy-7-methoxy-8-(3-(((S)-7-methoxy-5-oxo-2,3,5,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)propoxy)-5-oxo-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate(4)

EDCl.HCl (27 mg, 0.14 mmol) was added to a solution of 145 andAzido-PEG₈-acid (49 mg, 0.10 mmol) in CHCl₃ (6 mL) and stirred at roomtemperature for 1 hr. The solvent was evaporated under reduced pressure,to leave a yellow foam. Purification by prep HPLC gave the product 4 asan off-white solid (20 mg, 17% yield). LC/MS (method B): retention time6.01 min, (ES+) m/z 1320 [M+H]⁺.

(ii) (R)-2-((3-nitropyridin-2-yl)disulfaneyl)propyl(11S,11aS)-11-hydroxy-7-methoxy-8-(3-(((S)-7-methoxy-5-oxo-2,3,5,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)propoxy)-5-oxo-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate(5)

(a) tert-butyl(5-(3-(5-amino-4-((S)-2-(((tert-butyldimethylsilyl)oxy)methyl)pyrrolidine-1-carbonyl)-2-methoxyphenoxy)propoxy)-2-((S)-2-(((tert-butyldimethylsilyl)oxy)methyl)pyrrolidine-1-carbonyl)-4-methoxyphenyl)carbamate(I46)

Boc anhydride (0.5 g, 2.3 mmol, 1.0 eq) was added to a solution of 140(1.9 g, 2.3 mmol, 1.0 eq) in THF (50 mL) and stirred at 55° C. for 5 hr.The solvent was removed by evaporation under reduced pressure and theresidue purified by column chromatography (50-100% ethyl acetate/hexane)to leave the product as a yellow solid, 1.7 g (80%). LC/MS (method 1):rt 2.48 min, m/z (902.5) M+H.

(b) tert-butyl(2-((S)-2-(((tert-butyldimethylsilyl)oxy)methyl)pyrrolidine-1-carbonyl)-5-(3-(4-((S)-2-(((tert-butyldimethylsilyl)oxy)methyl)pyrrolidine-1-carbonyl)-2-methoxy-5-((((R)-2-((3-nitropyridin-2-yl)disulfaneyl)propoxy)carbonyl)amino)phenoxy)propoxy)-4-methoxyphenyl)carbamate(147)

Triphosgene (0.135 g, 0.455 mmol, 0.35 eq) was added to a solution of(2R)-2-[(3-nitro-2-pyridyl)disulfanyl]propan-1-ol (0.316 g, 1.28 mmol,1.05 eq) and pyridine (111 mg, 1.4 mmol, 1.15 eq) in anhydrousdichloromethane (5 mL) and stirred at room temperature for 30 min. Theresulting solution was then added to a solution of 146 (1.10 g, 1.22mmol, 1.0 eq) and pyridine (106 mg, 1.34 mmol, 1.1 eq) in anhydrousdichloromethane (10 mL) and stirred at room temperature for 60 min. Thesolvent was removed by evaporation under reduced pressure and theresidue purified by column chromatography (40-50% ethyl acetate/hexane)to leave the product as a yellow foam, 1.21 g (85%). LC/MS (method 1):rt 2.53 min, m/z (1174.5) M+H.

(c) tert-butyl(2-((S)-2-(hydroxymethyl)pyrrolidine-1-carbonyl)-5-(3-(4-((S)-2-(hydroxymethyl)pyrrolidine-1-carbonyl)-2-methoxy-5-((((R)-2-((3-nitropyridin-2-yl)disulfaneyl)propoxy)carbonyl)amino)phenoxy)propoxy)-4-methoxyphenyl)carbamate(I48)

I47 (1.21 g, 1.03 mmol) was dissolved in a mixture of acetic acid (5mL), THF (1 mL), methanol (1 mL) and water (2 mL). The resultingsolution was stirred at room temperature for 90 mins then evaporated todryness. The residue was taken up in ethyl acetate (50 mL), washed withwater (50 mL), then sat NaHCO₃ (50 mL), dried (MgSO₄) and evaporatedunder reduced pressure. The residue was purified by column (4%methanol/DCM) to leave the product as a yellow solid, 0.97 g (100%).LC/MS (method 1): rt 1.87 min, m/z (946.0) M+H.

(d) tert-butyl(11S,11aS)-11-hydroxy-8-(3-(((11S,11aS)-11-hydroxy-7-methoxy-10-(((R)-2-((3-nitropyridin-2-yl)disulfaneyl)propoxy)carbonyl)-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)propoxy)-7-methoxy-5-oxo-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate(I49)

Stahl Aerobic Oxidation TEMPO solution (2.05 mL, 0.4 mmol, 0.2 mol/L)followed by Tetrakisacetonitrile copper(I) triflate (0.15 g, 0.40 mmol)was added to a solution of 148 (0.97 g, 1.0 mmol) in DCM (20 mL, 312.0mmol). The resulting mixture was heated at 35° C. for 15 hrs. Theorganic phase was washed with water (25 mL), dried (biotage) andevaporated to dryness under reduced pressure and purified by columnchromatography (3-6% methanol/DCM) to leave the product as a whitesolid, 0.77 g (79%). LC/MS (method 1): rt 1.70 min, m/z (941.9) M+H.

(e) (R)-2-((3-nitropyridin-2-yl)disulfaneyl)propyl(11S,11aS)-11-hydroxy-7-methoxy-8-(3-(((S)-7-methoxy-5-oxo-2,3,5,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)propoxy)-5-oxo-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate(5)

Trifluoroacetic acid (4.5 mL) was added to water (0.5 mL) and cooled to0° C. This solution was then added to 149 (0.75 g, 0.80 mmol) and theresulting mixture stirred at 0° C. for 2 hrs. The solvent was removedunder reduced pressure, the residue taken up in DCM (10 mL) and thereaction mixture neutralised by the addition of sat NaHCO₃. After drying(biotage) and evaporation under reduced pressure, the residue waspurified by column (4-6% methanol/DCM) to leave the product as a brightyellow solid, 0.6 g (91%). LC/MS (method 2): rt 6.04 min, m/z (824.0)M+H.

Analytical LC/MS Conditions for Example 5(ii)

Positive mode electrospray mass spectrometry was performed using aWaters Aquity H-class SQD2. Mobile phases used were solvent A (waterwith 0.1% formic acid) and solvent B (acetonitrile with 0.1% formicacid).

Method 1:

Gradient for routine 3-minute run: Initial composition 5% B held over 25seconds, then increased from 5% B to 100% B over a 1 minute 35 seconds'period. The composition was held for 50 seconds at 100% B, then returnedto 5% B in 5 seconds and held there for 5 seconds. The total duration ofthe gradient run was 3.0 minutes. Flow rate was 0.8 mL/minute. Detectionwas at 254 nm. Column: Waters Acquity UPLC® BEH Shield RP18 1.7 μm2.1×50 mm at 50° C. fitted with Waters Acquity UPLC® BEH Shield RP18VanGuard Pre-column, 130A, 1.7 μm, 2.1 mm×5 mm.

Method 2:

Gradient for 15-minute run: Initial composition 5% B held over 1 minute,then increased from 5% B to 100% B over a 9 minute period. Thecomposition was held for 2 minutes at 100% B, then returned to 5% B in10 seconds and held there for 2 minutes 50 seconds. The total durationof the gradient run was 15.0 minutes. Flow rate was 0.8 mL/minute (for3-minute run) and 0.6 mL/minute (for 15-minute run). Detection was at254 nm. Column: ACE Excel 2 C18-AR, 2μ, 3.0×100 mm fitted with WatersAcquity UPLC® BEH Shield RP18 VanGuard Pre-column, 130A, 1.7 μm, 2.1mm×5 mm.

Example 6—Conjugation Conj-HER-1

A 50 mM solution of Dithiothreitol (DTT) in phosphate-buffered saline pH7.4 (PBS) was added (80 molar equivalent/antibody, 16 micromoles, 0.32mL at 50 mM) to a 12 mL solution of antibody Herceptin (30 mg, 0.2micromoles) in reduction buffer containing PBS and 1 mMethylenediaminetetraacetic acid (EDTA) and a final antibodyconcentration of 2.5 mg/mL. The reduction mixture was heated at +25° C.for 4 hours (or until full reduction observed by UHPLC) in an orbitalshaker with gentle (60 rpm) shaking. After cooling down to roomtemperature, the reduced antibody was buffer exchanged, via spin filterusing 50 KDa MWCO vivaspin, into a reoxidation buffer containing PBS pH7.4 and 1 mM EDTA to remove all the excess reducing agent. A 50 mMsolution of dehydroascorbic acid (DHAA, 15 molar equivalent/antibody, 3micromoles, 0.08 mL at 50 mM) in DMSO was added and the reoxidationmixture was allowed to react for 16 hours at room temperature withgentle (60 rpm) shaking at an antibody concentration of 1.5 mg/mL (oruntil full reoxidation of the cysteine thiols to reform the inter-chaincysteine disulfides is observed by UHPLC). The reoxidation mixture wascentrifuged for 3 min at 4000 rpm and then sterile-filtered using 0.22μm membrane filter. Compound 1 was added as a DMSO solution (10 molarequivalent/antibody, 1.0 micromoles, in 1.5 mL DMSO) to 13.5 mL of thisreoxidised antibody solution (15 mg, 0.1 micromoles) for a 10% (v/v)final DMSO concentration. The solution was shaken for 3 hours at +25° C.and then the conjugation was quenched with N-acetyl cysteine (15micromoles, 0.150 mL at 100 mM).

Excess free drug was removed via spin filter using 50 kDa MWCO vivaspininto buffer containing PBS pH 7.4. Extent of free drug removal wasmonitored by UHPLC-RP using neat conjugate. After complete removal offree drug, the ADC was filtered using 0.22 μm, Mustang filter understerile atmosphere and then stored at +4° C.

UHPLC analysis on a Shimadzu Prominence system using a Phenomenex Aeris3.6u XB-C18 150×2.1 mm column eluting with a gradient of water andacetonitrile on a reduced sample of Conj-HER-1 at 214 nm and 330 nm(Compound 1 specific) shows a mixture of light and heavy chains attachedto several molecules of Compound 1, consistent with a drug-per-antibodyratio (DAR) of 1.74 molecules of Compound 1 per antibody.

UHPLC analysis on a Shimadzu Prominence system using a Tosoh BioscienceTSKgel SuperSW mAb HTP 4 μm 4.6×150 mm column (with a 4 μm 3.0×20 mmguard column) eluting with 0.3 mL/minute sterile-filtered SEC buffercontaining 200 mM potassium phosphate pH 6.95, 250 mM potassium chlorideand 10% isopropanol (v/v) on a sample of Conj-HER-1 at 280 nm shows amonomer purity of greater than 99%. UHPLC SEC analysis gives aconcentration of final ADC at 1.39 mg/mL in 7.8 mL, obtained mass of ADCis 10.8 mg (72% yield).

Conj-HER-2

A 50 mM solution of Dithiothreitol (DTT) in phosphate-buffered saline pH7.4 (PBS) was added (80 molar equivalent/antibody, 55.5 micromoles, 1.11mL at 50 mM) to a 11.8 mL solution of antibody Herceptin (104 mg, 0.69micromoles) in reduction buffer containing PBS and 1 mMethylenediaminetetraacetic acid (EDTA) and a final antibodyconcentration of 4.0 mg/mL. The reduction mixture was heated at +25° C.for 3.5 hours (or until full reduction observed by UHPLC) in an orbitalshaker with gentle (60 rpm) shaking. After cooling down to roomtemperature, the reduced antibody was buffer exchanged, via spin filterusing 50 KDa MWCO vivaspin, into a reoxidation buffer containing PBS pH7.4 and 1 mM EDTA to remove all the excess reducing agent. A 50 mMsolution of dehydroascorbic acid (DHAA, 20 molar equivalent/antibody,12.4 micromoles, 0.25 mL at 50 mM) in DMSO was added and the reoxidationmixture was allowed to react for 16 hours at room temperature withgentle (60 rpm) shaking at an antibody concentration of 2.4 mg/mL (oruntil full reoxidation of the cysteine thiols to reform the inter-chaincysteine disulfides is observed by UHPLC). The reoxidation mixture wascentrifuged for 3 min at 4000 rpm and then sterile-filtered using 0.22μm membrane filter. Compound 2 was added as a DMSO solution (10 molarequivalent/antibody, 1.03 micromoles, in 1.40 mL DMSO) to 14 mL of thisreoxidised antibody solution (15.5 mg, 0.103 micromoles) for a 10% (v/v)final DMSO concentration. The solution was shaken for 1.5 hours at +25°C. and then the conjugation was quenched with N-acetyl cysteine (5.15micromoles, 0.051 mL at 100 mM).

Excess free drug was removed via Tangential Flow Filtration unit (TFF)using mPES, MidiKros® 30 kDa fiber filter with 115 cm² surface area,into buffer containing PBS pH 7.4. Extent of free drug removal wasmonitored by UHPLC-RP using neat conjugate. After complete removal offree drug, ADC was filtered using 0.22 μm, Mustang filter under sterileatmosphere and then stored at +4° C.

UHPLC analysis on a Shimadzu Prominence system using a Phenomenex Aeris3.6u XB-C18 150×2.1 mm column eluting with a gradient of water andacetonitrile on a reduced sample of Conj-HER-2 at 214 nm and 330 nm(Compound 2 specific) shows a mixture of light and heavy chains attachedto several molecules of Compound 2, consistent with a drug-per-antibodyratio (DAR) of 1.85 molecules of Compound 2 per antibody.

UHPLC analysis on a Shimadzu Prominence system using a Tosoh BioscienceTSKgel SuperSW mAb HTP 4 μm 4.6×150 mm column (with a 4 μm 3.0×20 mmguard column) eluting with 0.3 mL/minute sterile-filtered SEC buffercontaining 200 mM potassium phosphate pH 6.95, 250 mM potassium chlorideand 10% isopropanol (v/v) on a sample of Conj-HER-2 at 280 nm shows amonomer purity of greater than 98%. UHPLC SEC analysis gives aconcentration of final ADC at 0.88 mg/mL in 8.5 mL, obtained mass of ADCis 7.5 mg (48% yield).

Conj-HER-3

A 50 mM solution of tris(2-carboxyethyl)phosphine (TCEP) inphosphate-buffered saline pH 7.4 (PBS) was added (40 molarequivalent/antibody, 40 micromoles, 0.08 mL at 50 mM) to a 1.39 mLsolution of antibody Herceptin (15 mg, 0.1 micromoles) in reductionbuffer containing PBS and 1 mM ethylenediaminetetraacetic acid (EDTA)and a final antibody concentration of 4.0 mg/mL. The reduction mixturewas heated at +37° C. for 2 hours (or until full reduction observed byUHPLC) in an orbital shaker with gentle (60 rpm) shaking. After coolingdown to room temperature, the reduced antibody was buffer exchanged, viaDialysis using 50 KDa MWCO cassette, into a reoxidation buffercontaining PBS pH 7.4 and 1 mM EDTA to remove all the excess reducingagent for 16 hours at RT. A 50 mM solution of dehydroascorbic acid(DHAA, 25 molar equivalent/antibody, 2.5 micromoles, 0.04 mL at 50 mM)in DMSO was added and the reoxidation mixture was allowed to react for 2hours at room temperature with gentle (60 rpm) shaking at an antibodyconcentration of 1.5 mg/mL. Due to incomplete oxidation another 0.04 mLof 50 mM DHAA added and further shaken at room temperature for 2 h.After that full reoxidation of the cysteine thiols to reform theinter-chain cysteine disulfides is observed by UHPLC. The reoxidationmixture was centrifuged for 3 min at 4000 rpm and then sterile-filteredusing 0.22 μm membrane filter. Compound 3 was added as a DMSO solution(10 molar equivalent/antibody, 0.8 micromoles, in 1.1 mL DMSO) to 11 mLof this reoxidised antibody solution (12 mg, 0.08 micromoles) for a 10%(v/v) final DMSO concentration. The solution was shaken for 1 hours at+25° C. and then the conjugation was quenched with N-acetyl cysteine(3.2 micromoles, 0.032 mL at 100 mM).

Excess free drug was removed via spin filter using 50 kDa MWCO vivaspininto buffer containing PBS pH 7.4. Extent of free drug removal wasmonitored by UHPLC-RP using neat conjugate. After complete removal offree drug, ADC was filtered using 0.22 μm, Mustang filter under sterileatmosphere and then stored at +4° C.

UHPLC analysis on a Shimadzu Prominence system using a Phenomenex Aeris3.6u XB-C18 150×2.1 mm column eluting with a gradient of water andacetonitrile on a reduced sample of Conj-HER-3 at 214 nm and 330 nm(Compound 3 specific) shows a mixture of light and heavy chains attachedto several molecules of Compound 3, consistent with a drug-per-antibodyratio (DAR) of 1.78 molecules of Compound 3 per antibody.

UHPLC analysis on a Shimadzu Prominence system using a Tosoh BioscienceTSKgel SuperSW mAb HTP 4 μm 4.6×150 mm column (with a 4 μm 3.0×20 mmguard column) eluting with 0.3 mL/minute sterile-filtered SEC buffercontaining 200 mM potassium phosphate pH 6.95, 250 mM potassium chlorideand 10% isopropanol (v/v) on a sample of Conj-HER-3 at 280 nm shows amonomer purity of greater than 94%. UHPLC SEC analysis gives aconcentration of final ADC at 1.14 mg/mL in 8.2 mL, obtained mass of ADCis 9.3 mg (62% yield).

Conj-R347-1

A 50 mM solution of Dithiothreitol (DTT) in phosphate-buffered saline pH7.4 (PBS) was added (80 molar equivalent/antibody, 697 micromoles, 13.87mL at 50 mM) to a 44.36 mL solution of antibody R³⁴⁷ (1300 mg, 8.67micromoles) in reduction buffer containing PBS and 1 mMethylenediaminetetraacetic acid (EDTA) and a final antibodyconcentration of 5.0 mg/mL. The reduction mixture was heated at +25° C.for 3.5 hours (or until full reduction observed by UHPLC) in an orbitalshaker with gentle (60 rpm) shaking. After cooling down to roomtemperature, the reduced antibody was buffer exchanged, via TangentialFlow Filtration unit (TFF) using mPES, MidiKros® 30 kDa fiber filterwith 235 cm² surface area, into a reoxidation buffer containing PBS pH7.4 and 1 mM EDTA to remove all the excess reducing agent. The reducedantibody was centrifuged for 3 min at 4000 rpm and then filtered using0.22 μm membrane filter. A 50 mM solution of dehydroascorbic acid (DHAA,15 molar equivalent/antibody, 130 micromoles, 2.6 mL at 50 mM) in DMSOwas added and the reoxidation mixture was allowed to react for 16 hoursat room temperature with gentle (60 rpm) shaking at an antibodyconcentration of 5.0 mg/mL (or until full reoxidation of the cysteinethiols to reform the inter-chain cysteine disulfides is observed byUHPLC). The reoxidation mixture was centrifuged for 3 min at 4000 rpmand then sterile-filtered using 0.22 μm membrane filter. Compound 1 wasadded as a DMSO solution (10 molar equivalent/antibody, 86.7 micromoles,in 23.4 mL DMSO) to 330 mL of this reoxidised antibody solution (1300mg, 8.67 micromoles) for a 10% (v/v) final DMSO concentration. Thesolution was shaken for 3 hours at +25° C. and then the conjugation wasquenched with N-acetyl cysteine (433 micromoles, 4.33 mL at 100 mM).

Excess free drug was removed via Tangential Flow Filtration unit (TFF)using mPES, MidiKros® 30 kDa fiber filter with 235 cm² surface area,into buffer containing PBS pH 7.4. Extent of free drug removal wasmonitored by UHPLC-RP using neat conjugate. After complete removal offree drug, ADC was formulated onto 25 mM Histidine, 200 mM Sucrose, pH6.0. ADC was filtered using 0.22 μm, Mustang filter under sterileatmosphere and then stored at −78° C.

UHPLC analysis on a Shimadzu Prominence system using a Phenomenex Aeris3.6u XB-C18 150×2.1 mm column eluting with a gradient of water andacetonitrile on a reduced sample of Conj-R347-1 at 214 nm and 330 nm(Compound 1 specific) shows a mixture of light and heavy chains attachedto several molecules of Compound 1, consistent with a drug-per-antibodyratio (DAR) of 1.82 molecules of Compound 1 per antibody.

UHPLC analysis on a Shimadzu Prominence system using a Tosoh BioscienceTSKgel SuperSW mAb HTP 4 μm 4.6×150 mm column (with a 4 μm 3.0×20 mmguard column) eluting with 0.3 mL/minute sterile-filtered SEC buffercontaining 200 mM potassium phosphate pH 6.95, 250 mM potassium chlorideand 10% isopropanol (v/v) on a sample of ADC at 280 nm shows a monomerpurity of greater than 99%. UHPLC SEC analysis gives a concentration offinal Conj-R347-1 at 10.11 mg/mL in 113 mL, obtained mass of ADC is 1141mg (88% yield).

Conj-R347-2

A 50 mM solution of Dithiothreitol (DTT) in phosphate-buffered saline pH7.4 (PBS) was added (80 molar equivalent/antibody, 213 micromoles, 4.3mL at 50 mM) to a 13.7 mL solution of antibody R347 (400 mg, 2.67micromoles) in reduction buffer containing PBS and 1 mMethylenediaminetetraacetic acid (EDTA) and a final antibodyconcentration of 5.0 mg/mL. The reduction mixture was heated at +25° C.for 3.5 hours (or until full reduction observed by UHPLC) in an orbitalshaker with gentle (60 rpm) shaking. After cooling down to roomtemperature, the reduced antibody was buffer exchanged, via TangentialFlow Filtration unit (TFF) using mPES, MidiKros® 30 kDa fiber filterwith 115 cm² surface area, into a reoxidation buffer containing PBS pH7.4 and 1 mM EDTA to remove all the excess reducing agent. The reducedantibody was centrifuged for 3 min at 4000 rpm and then filtered using0.22 μm membrane filter. A 50 mM solution of dehydroascorbic acid (DHAA,15 molar equivalent/antibody, 40 micromoles, 0.8 mL at 50 mM) in DMSOwas added and the reoxidation mixture was allowed to react for 16 hoursat room temperature with gentle (60 rpm) shaking at an antibodyconcentration of 3.0 mg/mL (or until full reoxidation of the cysteinethiols to reform the inter-chain cysteine disulfides is observed byUHPLC). The reoxidation mixture was centrifuged for 3 min at 4000 rpmand then sterile-filtered using 0.22 μm membrane filter. Compound 2 wasadded as a DMSO solution (10 molar equivalent/antibody, 26.7 micromoles,in 15.5 mL DMSO) to 330 mL of this reoxidised antibody solution (400 mg,2.67 micromoles) for a 10% (v/v) final DMSO concentration. The solutionwas shaken for 1.5 hours at +25° C. and then the conjugation wasquenched with N-acetyl cysteine (125 micromoles, 1.25 mL at 100 mM).

Excess free drug was removed via Tangential Flow Filtration unit (TFF)using mPES, MidiKros® 30 kDa fiber filter with 115 cm² surface area,into buffer containing PBS pH 7.4. Extent of free drug removal wasmonitored by UHPLC-RP using neat conjugate. After complete removal offree drug, ADC was formulated onto 25 mM Histidine, 200 mM Sucrose, pH6.0. ADC was filtered using 0.22 μm, Mustang filter under sterileatmosphere and then stored at −78° C.

UHPLC analysis on a Shimadzu Prominence system using a Phenomenex Aeris3.6u XB-C18 150×2.1 mm column eluting with a gradient of water andacetonitrile on a reduced sample of Conj-R347-2 at 214 nm and 330 nm(Compound 2 specific) shows a mixture of light and heavy chains attachedto several molecules of Compound 2, consistent with a drug-per-antibodyratio (DAR) of 1.8 molecules of Compound 2 per antibody.

UHPLC analysis on a Shimadzu Prominence system using a Tosoh BioscienceTSKgel SuperSW mAb HTP 4 μm 4.6×150 mm column (with a 4 μm 3.0×20 mmguard column) eluting with 0.3 mL/minute sterile-filtered SEC buffercontaining 200 mM potassium phosphate pH 6.95, 250 mM potassium chlorideand 10% isopropanol (v/v) on a sample of Conj-R347-2 at 280 nm shows amonomer purity of greater than 99%. UHPLC SEC analysis gives aconcentration of final ADC at 3.06 mg/mL in 93 mL, obtained mass of ADCis 284 mg (71% yield).

Conj-R347-3

A 50 mM solution of Dithiothreitol (DTT) in phosphate-buffered saline pH7.4 (PBS) was added (80 molar equivalent/antibody, 240 micromoles, 4.8mL at 50 mM) to a 15.36 mL solution of antibody R347 (450 mg, 3.0micromoles) in reduction buffer containing PBS and 1 mMethylenediaminetetraacetic acid (EDTA) and a final antibodyconcentration of 5.0 mg/mL. The reduction mixture was heated at +25° C.for 3.5 hours (or until full reduction observed by UHPLC) in an orbitalshaker with gentle (60 rpm) shaking. After cooling down to roomtemperature, the reduced antibody was buffer exchanged, via TangentialFlow Filtration unit (TFF) using mPES, MidiKros® 30 kDa fiber filterwith 235 cm² surface area, into a reoxidation buffer containing PBS pH7.4 and 1 mM EDTA to remove all the excess reducing agent. The reducedantibody was centrifuged for 3 min at 4000 rpm and then filtered using0.22 μm membrane filter. A 50 mM solution of dehydroascorbic acid (DHAA,15 molar equivalent/antibody, 45 micromoles, 0.9 mL at 50 mM) in DMSOwas added and the reoxidation mixture was allowed to react for 16 hoursat room temperature with gentle (60 rpm) shaking at an antibodyconcentration of 3.5 mg/mL (or until full reoxidation of the cysteinethiols to reform the inter-chain cysteine disulfides is observed byUHPLC). The reoxidation mixture was centrifuged for 3 min at 4000 rpmand then sterile-filtered using 0.22 μm membrane filter. Compound 3 wasadded as a DMSO solution (10 molar equivalent/antibody, 30.0 micromoles,in 13.0 mL DMSO) to 330 mL of this reoxidised antibody solution (450 mg,3.0 micromoles) for a 10% (v/v) final DMSO concentration. The solutionwas shaken for 3 hours at +25° C. and then the conjugation was quenchedwith N-acetyl cysteine (150 micromoles, 1.5 mL at 100 mM).

Excess free drug was removed via Tangential Flow Filtration unit (TFF)using mPES, MidiKros® 30 kDa fiber filter with 235 cm² surface area,into buffer containing PBS pH 7.4. Extent of free drug removal wasmonitored by UHPLC-RP using neat conjugate. After complete removal offree drug, ADC was formulated onto 25 mM Histidine, 200 mM Sucrose, pH6.0. ADC was filtered using 0.22 μm, Mustang filter under sterileatmosphere and then stored at −78° C.

UHPLC analysis on a Shimadzu Prominence system using a Phenomenex Aeris3.6u XB-C18 150×2.1 mm column eluting with a gradient of water andacetonitrile on a reduced sample of Conjugate at 214 nm and 330 nm(Compound 3 specific) shows a mixture of light and heavy chains attachedto several molecules of Compound 3, consistent with a drug-per-antibodyratio (DAR) of 1.82 molecules of Compound 3 per antibody.

UHPLC analysis on a Shimadzu Prominence system using a Tosoh BioscienceTSKgel SuperSW mAb HTP 4 μm 4.6×150 mm column (with a 4 μm 3.0×20 mmguard column) eluting with 0.3 mL/minute sterile-filtered SEC buffercontaining 200 mM potassium phosphate pH 6.95, 250 mM potassium chlorideand 10% isopropanol (v/v) on a sample of Conj-R347-3 at 280 nm shows amonomer purity of greater than 99%. UHPLC SEC analysis gives aconcentration of final ADC at 2.35 mg/mL in 174 mL, obtained mass ofConj-R347-3 is 409 mg (91% yield).

Conj-HLL2-1

A 50 mM solution of Dithiothreitol (DTT) in phosphate-buffered saline pH7.4 (PBS) was added (80 molar equivalent/antibody, 53.3 micromoles, 1.07mL at 50 mM) to a 11.8 mL solution of antibody HLL2 (100 mg, 0.6micromoles) in reduction buffer containing PBS and 1 mMethylenediaminetetraacetic acid (EDTA) and a final antibodyconcentration of 5.0 mg/mL. The reduction mixture was heated at +25° C.for 3.5 hours (or until full reduction observed by UHPLC) in an orbitalshaker with gentle (60 rpm) shaking. After cooling down to roomtemperature, the reduced antibody was buffer exchanged, via spin filterusing 50 KDa MWCO vivaspin, into a reoxidation buffer containing PBS pH7.4 and 1 mM EDTA to remove all the excess reducing agent. A 50 mMsolution of dehydroascorbic acid (DHAA, 15 molar equivalent/antibody, 9micromoles, 0.18 mL at 50 mM) in DMSO was added and the reoxidationmixture was allowed to react for 16 hours at room temperature withgentle (60 rpm) shaking at an antibody concentration of 3.0 mg/mL (oruntil full reoxidation of the cysteine thiols to reform the inter-chaincysteine disulfides is observed by UHPLC). The reoxidation mixture wascentrifuged for 3 min at 4000 rpm and then sterile-filtered using 0.22μm membrane filter. Compound 1 was added as a DMSO solution (10 molarequivalent/antibody, 1.2 micromoles, in 0.58 mL DMSO) to 7 mL of thisreoxidised antibody solution (18 mg, 0.12 micromoles) for a 10% (v/v)final DMSO concentration. The solution was shaken for 3 hours at +25° C.and then the conjugation was quenched with N-acetyl cysteine (6micromoles, 0.06 mL at 100 mM).

Excess free drug was removed via spin filter using 50 kDa MWCO vivaspininto buffer containing PBS pH 7.4. Extent of free drug removal wasmonitored by UHPLC-RP using neat conjugate. After complete removal offree drug, ADC was filtered using 0.22 μm, Mustang filter under sterileatmosphere and then stored at +4° C.

UHPLC analysis on a Shimadzu Prominence system using a Phenomenex Aeris3.6u XB-C18 150×2.1 mm column eluting with a gradient of water andacetonitrile on a reduced sample of Conj-HLL2-1 at 214 nm and 330 nm(Compound 1 specific) shows a mixture of light and heavy chains attachedto several molecules of Compound 1, consistent with a drug-per-antibodyratio (DAR) of 1.74 molecules of Compound 1 per antibody.

UHPLC analysis on a Shimadzu Prominence system using a Tosoh BioscienceTSKgel SuperSW mAb HTP 4 μm 4.6×150 mm column (with a 4 μm 3.0×20 mmguard column) eluting with 0.3 mL/minute sterile-filtered SEC buffercontaining 200 mM potassium phosphate pH 6.95, 250 mM potassium chlorideand 10% isopropanol (v/v) on a sample of Conj-HLL2-1 at 280 nm shows amonomer purity of greater than 98%. UHPLC SEC analysis gives aconcentration of final ADC at 1.6 mg/mL in 7.5 mL, obtained mass of ADCis 12 mg (67% yield).

Conj-HLL2-2

A 50 mM solution of Dithiothreitol (DTT) in phosphate-buffered saline pH7.4 (PBS) was added (80 molar equivalent/antibody, 53.3 micromoles, 1.07mL at 50 mM) to a 11.8 mL solution of antibody HLL2 (100 mg, 0.6micromoles) in reduction buffer containing PBS and 1 mMethylenediaminetetraacetic acid (EDTA) and a final antibodyconcentration of 5.0 mg/mL. The reduction mixture was heated at +25° C.for 3.5 hours (or until full reduction observed by UHPLC) in an orbitalshaker with gentle (60 rpm) shaking. After cooling down to roomtemperature, the reduced antibody was buffer exchanged, via spin filterusing 50 KDa MWCO vivaspin, into a reoxidation buffer containing PBS pH7.4 and 1 mM EDTA to remove all the excess reducing agent. A 50 mMsolution of dehydroascorbic acid (DHAA, 15 molar equivalent/antibody, 9micromoles, 0.18 mL at 50 mM) in DMSO was added and the reoxidationmixture was allowed to react for 16 hours at room temperature withgentle (60 rpm) shaking at an antibody concentration of 3.0 mg/mL (oruntil full reoxidation of the cysteine thiols to reform the inter-chaincysteine disulfides is observed by UHPLC). The reoxidation mixture wascentrifuged for 3 min at 4000 rpm and then sterile-filtered using 0.22μm membrane filter. Compound 2 was added as a DMSO solution (10 molarequivalent/antibody, 1.2 micromoles, in 0.58 mL DMSO) to 7 mL of thisreoxidised antibody solution (18 mg, 0.12 micromoles) for a 10% (v/v)final DMSO concentration. The solution was shaken for 3 hours at +25° C.and then the conjugation was quenched with N-acetyl cysteine (6micromoles, 0.06 mL at 100 mM).

Excess free drug was removed via spin filter using 50 kDa MWCO vivaspininto buffer containing PBS pH 7.4. Extent of free drug removal wasmonitored by UHPLC-RP using neat conjugate. After complete removal offree drug, ADC was filtered using 0.22 μm, Mustang filter under sterileatmosphere and then stored at +4° C.

UHPLC analysis on a Shimadzu Prominence system using a Phenomenex Aeris3.6u XB-C18 150×2.1 mm column eluting with a gradient of water andacetonitrile on a reduced sample of Conjugate at 214 nm and 330 nm(Compound 2 specific) shows a mixture of light and heavy chains attachedto several molecules of Compound 2, consistent with a drug-per-antibodyratio (DAR) of 1.78 molecules of Compound 2 per antibody.

UHPLC analysis on a Shimadzu Prominence system using a Tosoh BioscienceTSKgel SuperSW mAb HTP 4 μm 4.6×150 mm column (with a 4 μm 3.0×20 mmguard column) eluting with 0.3 mL/minute sterile-filtered SEC buffercontaining 200 mM potassium phosphate pH 6.95, 250 mM potassium chlorideand 10% isopropanol (v/v) on a sample of Conj-HLL2-2 at 280 nm shows amonomer purity of greater than 98%. UHPLC SEC analysis gives aconcentration of final ADC at 1.56 mg/mL in 8.0 mL, obtained mass ofConj-HLL2-2 is 12.5 mg (69% yield).

Conj-HLL2-3

A 50 mM solution of Dithiothreitol (DTT) in phosphate-buffered saline pH7.4 (PBS) was added (80 molar equivalent/antibody, 53.3 micromoles, 1.07mL at 50 mM) to a 11.8 mL solution of antibody HLL2 (100 mg, 0.6micromoles) in reduction buffer containing PBS and 1 mMethylenediaminetetraacetic acid (EDTA) and a final antibodyconcentration of 5.0 mg/mL. The reduction mixture was heated at +25° C.for 3.5 hours (or until full reduction observed by UHPLC) in an orbitalshaker with gentle (60 rpm) shaking. After cooling down to roomtemperature, the reduced antibody was buffer exchanged, via spin filterusing 50 KDa MWCO vivaspin, into a reoxidation buffer containing PBS pH7.4 and 1 mM EDTA to remove all the excess reducing agent. A 50 mMsolution of dehydroascorbic acid (DHAA, 15 molar equivalent/antibody, 9micromoles, 0.18 mL at 50 mM) in DMSO was added and the reoxidationmixture was allowed to react for 16 hours at room temperature withgentle (60 rpm) shaking at an antibody concentration of 3.0 mg/mL (oruntil full reoxidation of the cysteine thiols to reform the inter-chaincysteine disulfides is observed by UHPLC). The reoxidation mixture wascentrifuged for 3 min at 4000 rpm and then sterile-filtered using 0.22μm membrane filter. Compound 3 was added as a DMSO solution (10 molarequivalent/antibody, 1.2 micromoles, in 0.58 mL DMSO) to 7 mL of thisreoxidised antibody solution (18 mg, 0.12 micromoles) for a 10% (v/v)final DMSO concentration. The solution was shaken for 3 hours at +25° C.and then the conjugation was quenched with N-acetyl cysteine (6micromoles, 0.06 mL at 100 mM).

Excess free drug was removed via spin filter using 50 kDa MWCO vivaspininto buffer containing PBS pH 7.4. Extent of free drug removal wasmonitored by UHPLC-RP using neat conjugate. After complete removal offree drug, ADC was filtered using 0.22 μm, Mustang filter under sterileatmosphere and then stored at +4° C.

UHPLC analysis on a Shimadzu Prominence system using a Phenomenex Aeris3.6u XB-C18 150×2.1 mm column eluting with a gradient of water andacetonitrile on a reduced sample of Conjugate at 214 nm and 330 nm(Compound 3 specific) shows a mixture of light and heavy chains attachedto several molecules of Compound 3, consistent with a drug-per-antibodyratio (DAR) of 1.79 molecules of Compound 3 per antibody.

UHPLC analysis on a Shimadzu Prominence system using a Tosoh BioscienceTSKgel SuperSW mAb HTP 4 μm 4.6×150 mm column (with a 4 μm 3.0×20 mmguard column) eluting with 0.3 mL/minute sterile-filtered SEC buffercontaining 200 mM potassium phosphate pH 6.95, 250 mM potassium chlorideand 10% isopropanol (v/v) on a sample of Conj-HLL2-3 at 280 nm shows amonomer purity of greater than 98%. UHPLC SEC analysis gives aconcentration of final ADC at 1.73 mg/mL in 8.2 mL, obtained mass ofConj-HLL2-3 is 14.2 mg (79% yield).

Conj-CD79b-1

A 50 mM solution of Dithiothreitol (DTT) in phosphate-buffered saline pH7.4 (PBS) was added (80 molar equivalent/antibody, 53.6 micromoles, 1.07mL at 50 mM) to a 13.9 mL solution of antibody CD79b (100 mg, 0.67micromoles) in reduction buffer containing PBS and 1 mMethylenediaminetetraacetic acid (EDTA) and a final antibodyconcentration of 4.0 mg/mL. The reduction mixture was heated at +25° C.for 4 hours (or until full reduction observed by UHPLC) in an orbitalshaker with gentle (60 rpm) shaking. After cooling down to roomtemperature, the reduced antibody was buffer exchanged, via spin filterusing 50 KDa MWCO vivaspin, into a reoxidation buffer containing PBS pH7.4 and 1 mM EDTA to remove all the excess reducing agent. A 50 mMsolution of dehydroascorbic acid (DHAA, 15 molar equivalent/antibody, 9micromoles, 0.18 mL at 50 mM) in DMSO was added and the reoxidationmixture was allowed to react for 16 hours at room temperature withgentle (60 rpm) shaking at an antibody concentration of 2.0 mg/mL (oruntil full reoxidation of the cysteine thiols to reform the inter-chaincysteine disulfides is observed by UHPLC). The reoxidation mixture wascentrifuged for 3 min at 4000 rpm and then sterile-filtered using 0.22μm membrane filter. Compound 1 was added as a DMSO solution (10 molarequivalent/antibody, 1.2 micromoles, in 1.0 mL DMSO) to 9.0 mL of thisreoxidised antibody solution (18 mg, 0.12 micromoles) for a 10% (v/v)final DMSO concentration. The solution was shaken for 2 hours at +25° C.and then the conjugation was quenched with N-acetyl cysteine (4.8micromoles, 0.048 mL at 100 mM).

Excess free drug was removed via spin filter using 50 kDa MWCO vivaspininto buffer containing PBS pH 7.4. Extent of free drug removal wasmonitored by UHPLC-RP using neat conjugate. After complete removal offree drug, ADC was filtered using 0.22 μm, Mustang filter under sterileatmosphere and then stored at +4° C.

UHPLC analysis on a Shimadzu Prominence system using a Phenomenex Aeris3.6u XB-C18 150×2.1 mm column eluting with a gradient of water andacetonitrile on a reduced sample of Conjugate at 214 nm and 330 nm(Compound 1 specific) shows a mixture of light and heavy chains attachedto several molecules of Compound 1, consistent with a drug-per-antibodyratio (DAR) of 1.90 molecules of Compound 1 per antibody.

UHPLC analysis on a Shimadzu Prominence system using a Tosoh BioscienceTSKgel SuperSW mAb HTP 4 μm 4.6×150 mm column (with a 4 μm 3.0×20 mmguard column) eluting with 0.3 mL/minute sterile-filtered SEC buffercontaining 200 mM potassium phosphate pH 6.95, 250 mM potassium chlorideand 10% isopropanol (v/v) on a sample of Conj-CD79b-1 at 280 nm shows amonomer purity of greater than 98%. UHPLC SEC analysis gives aconcentration of final ADC at 2.00 mg/mL in 7.85 mL, obtained mass ofConj-CD79b-1 is 15.7 mg (79% yield).

Conj-CD79b-2

A 50 mM solution of Dithiothreitol (DTT) in phosphate-buffered saline pH7.4 (PBS) was added (80 molar equivalent/antibody, 53.6 micromoles, 1.07mL at 50 mM) to a 13.9 mL solution of antibody CD79b (100 mg, 0.67micromoles) in reduction buffer containing PBS and 1 mMethylenediaminetetraacetic acid (EDTA) and a final antibodyconcentration of 4.0 mg/mL. The reduction mixture was heated at +25° C.for 4 hours (or until full reduction observed by UHPLC) in an orbitalshaker with gentle (60 rpm) shaking. After cooling down to roomtemperature, the reduced antibody was buffer exchanged, via spin filterusing 50 KDa MWCO vivaspin, into a reoxidation buffer containing PBS pH7.4 and 1 mM EDTA to remove all the excess reducing agent. A 50 mMsolution of dehydroascorbic acid (DHAA, 15 molar equivalent/antibody, 9micromoles, 0.18 mL at 50 mM) in DMSO was added and the reoxidationmixture was allowed to react for 16 hours at room temperature withgentle (60 rpm) shaking at an antibody concentration of 2.0 mg/mL (oruntil full reoxidation of the cysteine thiols to reform the inter-chaincysteine disulfides is observed by UHPLC). The reoxidation mixture wascentrifuged for 3 min at 4000 rpm and then sterile-filtered using 0.22μm membrane filter. Compound 2 was added as a DMSO solution (10 molarequivalent/antibody, 1.2 micromoles, in 1.0 mL DMSO) to 9.0 mL of thisreoxidised antibody solution (18 mg, 0.12 micromoles) for a 10% (v/v)final DMSO concentration. The solution was shaken for 2 hours at +25° C.and then the conjugation was quenched with N-acetyl cysteine (4.8micromoles, 0.048 mL at 100 mM).

Excess free drug was removed via spin filter using 50 kDa MWCO vivaspininto buffer containing PBS pH 7.4. Extent of free drug removal wasmonitored by UHPLC-RP using neat conjugate. After complete removal offree drug, ADC was filtered using 0.22 μm, Mustang filter under sterileatmosphere and then stored at +4° C.

UHPLC analysis on a Shimadzu Prominence system using a Phenomenex Aeris3.6u XB-C18 150×2.1 mm column eluting with a gradient of water andacetonitrile on a reduced sample of Conjugate at 214 nm and 330 nm(Compound 2 specific) shows a mixture of light and heavy chains attachedto several molecules of Compound 2, consistent with a drug-per-antibodyratio (DAR) of 1.87 molecules of Compound 2 per antibody. UHPLC analysison a Shimadzu Prominence system using a Tosoh Bioscience TSKgel SuperSWmAb HTP 4 μm 4.6×150 mm column (with a 4 μm 3.0×20 mm guard column)eluting with 0.3 mL/minute sterile-filtered SEC buffer containing 200 mMpotassium phosphate pH 6.95, 250 mM potassium chloride and 10%isopropanol (v/v) on a sample of Conj-CD79b-2 at 280 nm shows a monomerpurity of greater than 98%. UHPLC SEC analysis gives a concentration offinal ADC at 2.25 mg/mL in 5.9 mL, obtained mass of Conj-CD79b-2 is 13.3mg (66% yield).

Conj-1C1-1

A 50 mM solution of Dithiothreitol (DTT) in phosphate-buffered saline pH7.4 (PBS) was added (80 molar equivalent/antibody, 75 micromoles, 1.5 mLat 50 mM) to a 26.4 mL solution of antibody 1C1 (140 mg, 0.93micromoles) in reduction buffer containing PBS and 1 mMethylenediaminetetraacetic acid (EDTA) and a final antibodyconcentration of 5.0 mg/mL. The reduction mixture was heated at +25° C.for 3.5 hours (or until full reduction observed by UHPLC) in an orbitalshaker with gentle (60 rpm) shaking. After cooling down to roomtemperature, the reduced antibody was buffer exchanged, via spin filterusing 50 KDa MWCO vivaspin, into a reoxidation buffer containing PBS pH7.4 and 1 mM EDTA to remove all the excess reducing agent. A 50 mMsolution of dehydroascorbic acid (DHAA, 15 molar equivalent/antibody, 14micromoles, 0.28 mL at 50 mM) in DMSO was added and the reoxidationmixture was allowed to react for 16 hours at room temperature withgentle (60 rpm) shaking at an antibody concentration of 3.0 mg/mL (oruntil full reoxidation of the cysteine thiols to reform the inter-chaincysteine disulfides is observed by UHPLC). The reoxidation mixture wascentrifuged for 3 min at 4000 rpm and then sterile-filtered using 0.22μm membrane filter. Compound 1 was added as a DMSO solution (10 molarequivalent/antibody, 1.3 micromoles, in 0.6 mL DMSO) to 6 mL of thisreoxidised antibody solution (20 mg, 0.133 micromoles) for a 10% (v/v)final DMSO concentration. The solution was shaken for 4 hours at +25° C.and then the conjugation was quenched with N-acetyl cysteine (6.65micromoles, 0.067 mL at 100 mM).

Excess free drug was removed via spin filter using 50 kDa MWCO vivaspininto buffer containing PBS pH 7.4. Extent of free drug removal wasmonitored by UHPLC-RP using neat conjugate. After complete removal offree drug, buffer exchanged onto 25 mM Histidine, 200 mM Sucrose, pH6.0. ADC was filtered using 0.22 μm, Mustang filter under sterileatmosphere and then stored at −78° C.

UHPLC analysis on a Shimadzu Prominence system using a Phenomenex Aeris3.6u XB-C18 150×2.1 mm column eluting with a gradient of water andacetonitrile on a reduced sample of Conjugate at 214 nm and 330 nm(Compound 1 specific) shows a mixture of light and heavy chains attachedto several molecules of Compound 1, consistent with a drug-per-antibodyratio (DAR) of 1.86 molecules of Compound 1 per antibody.

UHPLC analysis on a Shimadzu Prominence system using a Tosoh BioscienceTSKgel SuperSW mAb HTP 4 μm 4.6×150 mm column (with a 4 μm 3.0×20 mmguard column) eluting with 0.3 mL/minute sterile-filtered SEC buffercontaining 200 mM potassium phosphate pH 6.95, 250 mM potassium chlorideand 10% isopropanol (v/v) on a sample of ADC at 280 nm shows a monomerpurity of greater than 98%. UHPLC SEC analysis gives a concentration offinal ADC at 1.49 mg/mL in 8.0 mL, obtained mass of ADC is 11.9 mg (60%yield).

Conj-HER-1++ (High DAR)

A 50 mM solution of Dithiothreitol (DTT) in phosphate-buffered saline pH7.4 (PBS) was added (100 molar equivalent/antibody, 6.7 micromoles, 0.13mL at 50 mM) to a 5 mL solution of antibody (10 mg, 67 nanomoles) inreduction buffer containing PBS and 1 mM ethylenediaminetetraacetic acid(EDTA) and a final antibody concentration of 2.0 mg/mL. The resultantmixture was incubated at +25° C. for overnight (or until full reductionobserved by UHPLC) in an orbital shaker with gentle (60 rpm) shaking.After cooling down to room temperature, the reduced antibody was bufferexchanged, via spin filter using 50 KDa MWCO vivaspin, into conjugationbuffer containing PBS pH 7.4 and 1 mM EDTA to remove all the excessreducing agent. Compound 1 was added as a DMSO solution (20 molarequivalent/antibody, 1.34 micromoles, in 0.4 mL DMSO) to 4.0 mL of thisreduced antibody solution (10 mg, 67 nanomoles) for a 10% (v/v) finalDMSO concentration. The solution was shaken for 1 hour at +25° C. andthen conjugation was quenched with excess N-acetyl cysteine (6.7micromoles, 67 μL at 100 mM).

Resultant ADC was purified via preparative size exclusion column (GESephadex 26/60) fitted to an AKTA Start instrument using PBS, pH 7.4buffer. Fractions were collected and analysed for the monomeric contentusing Shimadzu Prominence system using a Tosoh Bioscience TSKgel SuperSWmAb HTP 4 μm 4.6×150 mm column (with a 4 μm 3.0×20 mm guard column)eluting with 0.3 mL/minute sterile-filtered SEC buffer containing 200 mMpotassium phosphate pH 6.95, 250 mM potassium chloride and 10%isopropanol (v/v) at 280 nm. Fractions with monomer content >92% werepooled and then concentrated using 50 kDa MWCO vivaspin into buffercontaining PBS pH 7.4. Extent of free drug removal was monitored byUHPLC-RP using neat conjugate and after complete removal of free drug,ADC was filtered using 0.22 μm, Mustang filter under sterile atmosphereand then stored at +4° C.

UHPLC analysis on a Shimadzu Prominence system using a Phenomenex Aeris3.6u XB-C18 150×2.1 mm column eluting with a gradient of water andacetonitrile on a reduced sample of Conjugate at 214 nm and 330 nm(Compound 1 specific) shows a mixture of light and heavy chains attachedto several molecules of Compound 1, consistent with a drug-per-antibodyratio (DAR) of 7.41 molecules of Compound 1 per antibody.

UHPLC analysis on a Shimadzu Prominence system using a Tosoh BioscienceTSKgel SuperSW mAb HTP 4 μm 4.6×150 mm column (with a 4 μm 3.0×20 mmguard column) eluting with 0.3 mL/minute sterile-filtered SEC buffercontaining 200 mM potassium phosphate pH 6.95, 250 mM potassium chlorideand 10% isopropanol (v/v) on a sample of ADC at 280 nm shows a monomerpurity of 95%. UHPLC SEC analysis gives a concentration of final ADC at1.44 mg/mL in 4.5 mL, obtained mass of ADC is 6.5 mg (65% yield).

Conj-HER-1+ (Medium DAR)

A 10 mM solution of tris(2-carboxyethyl)phosphine (TCEP) inphosphate-buffered saline pH 7.4 (PBS) was added (2 molarequivalent/antibody, 0.134 micromoles, 13.3 μL at 10 mM) to a 4 mLsolution of antibody (10 mg, 67 nanomoles) in reduction buffercontaining PBS and 1 mM ethylenediaminetetraacetic acid (EDTA) and afinal antibody concentration of 2.5 mg/mL. The reduction mixture washeated at +37° C. for 2 hours in an orbital shaker with gentle (60 rpm)shaking. Compound 1 was added as a DMSO solution (15 molarequivalent/antibody, 1.0 micromoles, 0.1 mL of 10 mM in 0.3 mL DMSO) andthe resultant mixture was shaken for 1.5 hours at +25° C. and then theconjugation was quenched with N-acetyl cysteine (5 micromoles, 50 μL at100 mM).

Excess free drug was removed via spin filter using 50 kDa MWCO vivaspininto buffer containing PBS pH 7.4. Extent of free drug removal wasmonitored by UHPLC-RP using neat conjugate. After complete removal offree drug, ADC was filtered using 0.22 μm, Mustang filter under sterileatmosphere and then stored at +4° C.

UHPLC analysis on a Shimadzu Prominence system using a Phenomenex Aeris3.6u XB-C18 150×2.1 mm column eluting with a gradient of water andacetonitrile on a reduced sample of Conjugate at 214 nm and 330 nm(Compound 1 specific) shows a mixture of light and heavy chains attachedto several molecules of Compound 1, consistent with a drug-per-antibodyratio (DAR) of 4.2 molecules of Compound 1 per antibody.

UHPLC analysis on a Shimadzu Prominence system using a Tosoh BioscienceTSKgel SuperSW mAb HTP 4 μm 4.6×150 mm column (with a 4 μm 3.0×20 mmguard column) eluting with 0.3 mL/minute sterile-filtered SEC buffercontaining 200 mM potassium phosphate pH 6.95, 250 mM potassium chlorideand 10% isopropanol (v/v) on a sample of ADC at 280 nm shows a monomerpurity of greater than 99%. UHPLC SEC analysis gives a concentration offinal ADC at 2.05 mg/mL in 3.6 mL, obtained mass of ADC is 7.36 mg (74%yield).

Example 7—In Vitro Testing MTS Cytotoxicity Method

The concentration and viability of cells from a sub-confluent (80-90%confluency) T75 flask are measured by trypan blue staining, and countedusing the LUNA-II™ Automated Cell Counter. Cells were diluted to2×10⁵/ml, dispensed (50 μl per well) into 96-well flat-bottom plates.

A stock solution (1 ml) of the antibody drug conjugate (ADC) to betested (20 μg/ml) was made by dilution of filter-sterilised ADC intocell culture medium. A set of 8×10-fold dilutions of stock ADC were madein a 24-well plate by serial transfer of 100 μl into 900 μl of cellculture medium. ADC dilution was dispensed (50 μl per well) into 4replicate wells of the 96-well plate, containing 50 μl cell suspensionseeded the previously. Control wells received 50 μl cell culture medium.The 96-well plate containing cells and ADCs was incubated at 37° C. in aCO₂-gassed incubator for the exposure time.

At the end of the incubation period, cell viability was measured by MTSassay. MTS (Promega) was dispensed (20 μl per well) into each well andincubated for 4 hours at 37° C. in the CO₂-gassed incubator. Wellabsorbance was measured at 490 nm. Percentage cell survival wascalculated from the mean absorbance in the 4 ADC-treated wells comparedto the mean absorbance in the 4 control untreated wells (100%). IC₅₀ wasdetermined from the dose-response data using GraphPad Prism using thenon-linear curve fit algorithm: sigmoidal dose-response curve withvariable slope.

ADC incubation times were 4 days with SK-BR-3, MDA-MB-468, WSU-DLCL2,and SU-DHL-4, 5 days for Granta-519, 6 days for BJAB and 7 days forNCI-N87. MDA-MB-468, NCI-N87, WSU-DLCL2 and SU-DHL-4 were cultured inRPMI 1640 with Glutamax+10% (v/v) HyClone™ Fetal Bovine Serum,Granta-519 in DMEM+Glutamax with 10% (v/v) HyClone™ Fetal Bovine Serum,SK-BR-3 in McCoys 5A with 10% (v/v) HyClone™ Fetal Bovine Serum and BJABin RPMI 1640+Glutamax with 20% (v/v) HyClone™ Fetal Bovine Serum.

CellTiter-Glo Cytotoxicity Method

The concentration and viability of cells from a sub-confluent (80-90%confluency) T75 flask are measured by trypan blue staining, and countedusing the LUNA-II™ Automated Cell Counter. Cells were diluted and platedat 1500 cells per well (50 μl suspension per well) into white 96-wellflat-bottom plates.

A stock solution (1 ml) of antibody drug conjugate (ADC) to be tested(20 μg per ml) was made by dilution of filter-sterilised ADC into cellculture medium. A set of 8×10-fold dilutions of stock ADC were made in a24-well plate by serial transfer of 100 μl into 900 μl of cell culturemedium. ADC dilution was dispensed (50 μl per well) into 4 replicatewells of the 96-well plate, containing 50 μl cell suspension seeded thepreviously. Control wells received 50 μl cell culture medium. The96-well plate containing cells and ADCs was incubated at 37° C. in aCO₂-gassed incubator for the exposure time.

At the end of the incubation period, cell viability was measured byCellTiter-Glo assay. CellTiter-Glo (Promega) was dispensed at 100 μl perwell and shaken for 2 min before 10 min stabilisation at roomtemperature. Luminescence of each well was read. Percentage cellsurvival was calculated from the mean absorbance in the 4 ADC-treatedwells compared to the mean absorbance in the 4 control untreated wells(100%). IC₅₀ was determined from the dose-response data using GraphPadPrism using the non-linear curve fit algorithm: sigmoidal dose-responsecurve with variable slope.

ADC incubation time for PC-3 is 6 days. PC-3 were cultured in RPMI 1640with Glutamax+10% (v/v) HyClone™ Fetal Bovine Serum.

Results

GRANTA- EC₅₀ (μg/mL) SU-DHL-4 519 BJAB WSU-DLCL2 Conj-CD79b-1 1.21649.69 0.2211 >100 Conj-CD79b-2 0.4781 0.1124 0.005712 0.7559 Conj-R347-2>10

The cell lines SU-DHL-4, GRANTA519, BJAB and WSU-DL-CL2 all expressCD79b.

EC₅₀ (μg/mL) SK-BR-3 NCI-N87 MDA-MB-468 Conj-Her-1 0.06953 0.9677 3.492Conj-Her-3 0.0175 0.02502 2.322 Conj-Her-2 0.07427 0.08078 0.9533Conj-HER-1++ 0.1108 12.18 Conj-HER-1+ 0.3226 >10

The cell lines SK-BR-3 and NCI-N87 express Her2. The cell lineMDA-MB-468 does not express HER2.

EC₅₀ (μg/mL) PC-3 Conj-1C1-1 0.5408 Conj-R347-1 >100

Example 8—In Vivo Assays (i) Daudi

Conjugates tested: Conj-HLL2-1; Conj-HLL2-2; Conj-HLL2-3

Female CB.17 SCID mice, aged ten weeks, were injected with 0.1 ml of1×10⁷ Daudi cells subcutaneously in the right flank. When tumoursreached an average size of 100-150 mm³, treatment began. Mice wereweighed twice a week. Tumour size was measured twice a week. Animalswere monitored individually. The endpoint of the experiment was a tumourvolume of 1500 mm³ or 60 days, whichever came first.

Groups of 10 xenografted mice were injected i.v. with 0.2 ml per 20 g ofbody weight of antibody drug conjugate (ADC) in phosphate bufferedsaline (vehicle) or with 0.2 ml per 20 g of body weight of vehiclealone.

The concentration of ADC was adjusted to give the following doses:

Conjuagte Doses (mg ADC/kg body eight) Conj-HLL2-1 0.6 or 1.8Conj-HLL2-2 0.3 or 1 Conj-HLL2-3 0.1 or 0.3

All regimens were acceptably tolerated with little body weight loss.

Conj-HLL2-1:

The median time to end point (TTE) for vehicle-treated controls was 27.8days, establishing a maximum possible tumour growth delay (TGD) of 32.2days (116%) for the 60-day study. One non-treatment related death wasrecorded on day 28, and this animal was excluded from the analysis.

The 0.6 mg/kg regimen resulted in a TGD of 48.1 days (73%), which wasstatistically significant from controls (p<0.001). Furthermore, thisregimen had five of nine 59-regression responses consisting of twopartial and three complete regressions. Six animals attained the 1500mm3 endpoint, leaving 3 survivors after 60 days. The three survivors hada mean tumour volume of 221 mm³. There was one non-treatment relateddeath and this animal was excluded from the analysis.

The 1.8 mg/kg regimen resulted in the maximum possible, significant TGD(vs controls, p<0.001), had eight of ten 60-day survivors and produced asurvival benefit that was statistically significantly different fromvehicle-treated controls (p<0.001). Eight animals showed completeregression responses. Five of these animals were tumour free after 60days.

Both treatment regimens produced statistically significant survivalbenefits compared to control (p<0.001 for both 0.6 and 1.8 mg/ml).

Conj-HLL2-2:

The median time to end point (TTE) for vehicle-treated controls was 27.8days, establishing a maximum possible tumour growth delay (TGD) of 32.2days (116%) for the 60-day study.

The 0.3 and 1 mg/kg regimens both resulted in maximum achievable TGDs(32.2 days, 116%). Both of these results were statistically significantfrom controls (p<0.001 for each regimen).

Eighty percent of 0.3 mg/kg treated animals evinced regression responsesconsisting of two partial responses and six complete responses, four ofwhich remained tumour free at the study end. Four 0.3 mg/kg treatedanimals reached the tumour volume endpoint leaving six study survivorstumour free at the end of the study.

One hundred percent of 1 mg/kg treated animals showed regressionresponses and all animals were tumour free on day 60.

Both treatment regimens resulted in significant overall survivaldifferences versus controls (controls versus either 0.3 or 1 mg/kg,p<0.0001).

Conj-HLL2-3:

The median time to end point (TTE) for vehicle-treated controls was 27.8days, establishing a maximum possible tumour growth delay (TGD) of 32.2days (116%) for the 60-day study.

The 0.1 and 0.3 mg/kg regimens resulted in TGDs of 12.9 days (46%) and24.6 days (88%). Both of these results were statistically significantfrom controls (p<0.001 for each regimen).

Thirty percent regression responses were observed in animals treatedwith the 0.1 mg/ml regimen with three partial responses. All the animalsin this group reached the tumour volume endpoint by day 60.

Seventy percent of 0.3 mg/kg treated animals evinced regressionresponses consisting of three partial responses and four completeresponses, one of which remained tumour free at the study end. Four 0.3mg/kg treated animals reached the tumour volume endpoint leaving sixstudy survivors tumour free at the end of the study. Seven animals inthis group reached the tumour volume endpoint leaving three 60 daysurvivors with an MTV of 750 mm³ at study end.

Both treatment regimens resulted in significant overall survivaldifferences versus controls (controls versus either 0.1 or 0.13 mg/kg,p<0.0001).

(ii) JIMT-1

Conjugate tested: Conj-Her-3

Female CB.17 SCID mice, aged 10 weeks, were injected with 0.1 ml of1×10⁷ JIMT-1 cells in 50% Matrigel subcutaneously in the right flank.When tumours reached an average size of 100-150 mm³, treatment began.Mice were weighed twice a week. Tumour size was measured twice a week.Animals were monitored individually. The endpoint of the experiment wasa tumour volume of 1000 mm³ or 59 days, whichever came first.

Groups of 10 xenografted mice were injected i.v. with 0.2 ml per 20 g ofbody weight of antibody drug conjugate (ADC) in phosphate bufferedsaline (vehicle) or with 0.2 ml per 20 g of body weight of vehiclealone. The concentration of ADC was adjusted to give 1 or 3 mg ADC/kgbody weight in a single dose.

All regimens were acceptably tolerated with little body weight loss. Themedian time to end point (TTE) for vehicle-treated controls was 48.4days, establishing a maximum possible tumour growth delay (TGD) of 10.6days (22%) for the 59-day study.

A dose dependent effect was observed where in animals that received the1 mg/kg dose the median tumour volume remained static until day 34, thenprogressed thereafter, while for animals treated with 3 mg/kg there wasa small reduction in tumour size to an MTV of 81 mm³. Both dosingregimens resulted in a maximal TGD of 10.6 days (22%) versus the controlgroup (p<0.001 for both treated groups).

The 1 mg/kg regimen resulted in nine study survivors with an MTV of 650mm³ and no objective regression responses.

The 3 mg/kg regimen resulted in nine study survivors with 20% objectiveregression responses consisting of two partial responses. The MTV of thestudy survivors was 108 mm³. The treatment regimens were notsignificantly different from each other (p>0.05).

Both treatment regimens resulted in significant overall survivaldifferences versus controls (controls versus either 1 or 3 mg/kg,p<0.0001).

(iii) NCI-N87

Conjugate tested: Conj-Her-3

Female CB.17 SCID mice, aged ten weeks, were injected with 0.1 ml of1×10⁷ NCI-N87 cells in 50% Matrigel subcutaneously in the right flank.When tumours reached an average size of 100-150 mm³, treatment began.Mice were weighed twice a week. Tumour size was measured twice a week.Animals were monitored individually. The endpoint of the experiment wasa tumour volume of 800 mm³ or 81 days, whichever came first.

Groups of 10 xenografted mice were injected i.v. with 0.2 ml per 20 g ofbody weight of antibody drug conjugate (ADC) in phosphate bufferedsaline (vehicle) or with 0.2 ml per 20 g of body weight of vehiclealone. The concentration of ADC was adjusted to give 0.3 or 1 mg ADC/kgbody weight in a single dose.

All regimens were acceptably tolerated with little body weight loss. Sixof ten control tumours reached the 800 mm³, with time to endpoints (TTE)ranging 36.8 to 81.0 days. The median time to end point (TTE) forvehicle-treated controls was 77 days, establishing a maximum possibletumour growth delay (TGD) of 4 days (5%) for the 81-day study. Fourcontrol animals survived with a median tumour volume (MTV) of 696 mm³.

The 0.3 and 1 mg/kg regimens resulted in TGDs of 0.5 (1%) and 4.0 days(5%) respectively. Both of these results were not statisticallysignificant from controls, not each other (p>0.05). There were noobjective regressions recorded in either group. Five 0.3 mg/kg and seven1 mg/kg treated animals survived with MTVs of 550 mm³ in both groups.

Neither treatment regimen resulted in statistically significant survivalbenefits compared to control (p>0.05 for both treatment groups), andthere was no significant difference between the treatment groups(p>0.05).

(iv) NCI-N87

Conjugates tested: Conj-Her-1, Conj-Her-3

Female CB.17 SCID mice, aged eight weeks, were injected with 0.1 ml of1×10⁷ NCI-N87 in 50% Matrigel cells subcutaneously in the right flank.When tumours reached an average size of 100-150 mm³, treatment began.Mice were weighed twice a week. Tumour size was measured twice a week.Animals were monitored individually. The endpoint of the experiment wasa tumour volume of 800 mm³ or 78 days, whichever came first.

Groups of 10 xenografted mice were injected i.v. with 0.2 ml per 20 g ofbody weight of antibody drug conjugate (ADC) in phosphate bufferedsaline (vehicle) or with 0.2 ml per 20 g of body weight of vehiclealone.

The concentration of ADC was adjusted to give the following doses:

Conjugate Doses (mg ADC/kg body eight) Conj-Her-1 1, 3, or 6 Conj-Her-30.3, 1 and 3

All regimens were acceptably tolerated with little body weight loss.

Conj-Her-1:

The median time to end point (TTE) for vehicle-treated controls was 42days, establishing a maximum possible tumour growth delay (TGD) of 36days (86%) for the 60-day study.

The 0.6 and 1 mg/kg regimens resulted in TGDs of 9.9 (24%) and 11.6 days(28%) respectively. Neither of these results were statisticallysignificant from controls (p>0.05). All animals in both groups attainedthe 800 mm³ endpoint.

The 6 mg/kg regimen resulted in the maximum possible, significant TGD(vs controls, p<0.001), had eight of ten 60-day survivors and produced asurvival benefit that was statistically significantly different fromvehicle-treated controls (p<0.0001). One animal reached the 800 mm³endpoint at day 78, leaving nine survivors with mean tumour volumes of550 mm³.

There were no observable regression responses with 1, 3, or 6 mg/kgregimens.

Conj-Her-3:

The median time to end point (TTE) for vehicle-treated controls was 42.0days, establishing a maximum possible tumour growth delay (TGD) of 36.0days (86%) for the 78-day study.

The TGDs for 0.3, 1 and 3 mg/kg were 15.1 (36%), 30.6 (73%) and 36.0(86%) days respectively. There were significant differences for 1 and 3mg/kg versus controls (p<0.001 for both treatment groups, but not 0.3mg/kg (p>0.05). No regression responses were observed in animals treatedwith 0.3 and 1 mg/kg ADC. Ninety percent regression responses wereobserved in 3 mg/kg treated animals. This consisted of eight partialresponses and one complete response, which remained tumour free at studyend. All animals treated with 0.3 mg/kg reached the 800 mm³ endpoint.Five 1 mg/kg treated animals attained endpoint leaving five 78 daysurvivors. These had an MTV of 486 mm³. All ten 3 mg/kg treated animalssurvived the study with an MTV of 63 mm³.

The 1 and 3 mg/kg regimens resulted in significant survival differencesversus controls (p<0.001 for both treatment groups). The 0.3 mg/kgtreatment group was not statistically significant from control (p>0.05).Both 1 and 3 mg/kg treatment were significantly different from the 0.3mg/kg group (p<0.001 and p<0.0001 respectively).

(v) NCI-N87

Conjugate tested: Conj-Her-2

Female CB.17 SCID mice, aged eight weeks, were injected with 0.1 ml of1×10⁷ NCI-N87 cells in 50% Matrigel subcutaneously in the right flank.When tumours reached an average size of 100-150 mm³, treatment began.Mice were weighed twice a week. Tumour size was measured twice a week.Animals were monitored individually. The endpoint of the experiment wasa tumour volume of 800 mm³ or 79 days, whichever came first.

Groups of 10 xenografted mice were injected i.v. with 0.2 ml per 20 g ofbody weight of antibody drug conjugate (ADC) in phosphate bufferedsaline (vehicle) or with 0.2 ml per 20 g of body weight of vehiclealone. The concentration of ADC was adjusted to give 1 or 2 mg ADC/kgbody weight in a single dose.

All regimens were acceptably tolerated with little body weight loss. Themedian time to end point (TTE) for vehicle-treated controls was 49.6days, establishing a maximum possible tumour growth delay (TGD) of 29.4days (59%) for the 79-day study.

The 1 and 2 mg/kg regimens resulted in TGDs of 7.6 (15%) and 23.6 days(48%) respectively. Both of these results were statistically significantfrom controls (p<0.05 and p<0.001 respectively).

Eight animals treated with the 1 mg/kg regimen attained the 800 mm³endpoint leaving two 79-day survivors with an MTV of 694 mm³. Sevenanimals in the 2 mg/ml treated group reached the tumour volume endpointby day 79 leaving three end of study survivors with an MTV of 600 mm³.

Both treatment regimens resulted in significant overall survivaldifferences versus controls (controls versus 1 mg/kg, p<0.05; controlsversus 2 mg/kg, p<0.001).

Regression responses were not recorded with either regimen.

(vi) NCI-N87

Conjugates tested: Conj-Her-1, Conj-Her-1++

Female CB.17 SCID mice, aged eight weeks, were injected with 0.1 ml of1×10⁷ NCI-N87 in 50% Matrigel cells subcutaneously in the right flank.When tumours reached an average size of 100-150 mm³, treatment began.Mice were weighed twice a week. Tumour size was measured twice a week.Animals were monitored individually. The endpoint of the experiment wasa tumour volume of 800 mm³ or 81 days, whichever came first.

Groups of 10 xenografted mice were injected i.v. with 0.2 ml per 20 g ofbody weight of antibody drug conjugate (ADC) in phosphate bufferedsaline (vehicle) or with 0.2 ml per 20 g of body weight of vehiclealone.

The concentration of ADC was adjusted to give the following doses:

Conjugate Doses (mg ADC/kg body weight) Conj-Her-1 6, 18, 6 (qwk × 3), 8(qwk × 3) Conj-Her-1++ 6

All regimens were acceptably tolerated with little body weight loss.

The median time to end point (TTE) for vehicle-treated controls was 40.2days, establishing a maximum possible tumour growth delay (TGD) of 40days (86%) for the 80-day study.

Conj-Her-1:

The 6 and 18 mg/kg single-dose regimens resulted in the maximum possibletumor growth delays. In the 6 mg/kg single-dose regimen the mean tumourvolume for 5 mice was 600 mm³, with no observable regression responses.In the 18 mg/kg single-dose regimen there were ten survivors with a meantumour volume of 36 mm³. There were 4 partial regressions and 6 completeregressions.

The 6 and 8 mg/kg three weekly dose regimens also resulted in the in themaximum possible tumor growth delays. In the 6 mg/kg three weekly doseregimen the mean tumour volume for 9 mice at the end of the study was245 mm³, with two partial regressions. In the 8 mg/kg three weekly doseregimen there were ten survivors with a mean tumour volume of 92 mm³.There were 7 partial regressions, 3 complete regressions and 2 tumourfree survivors.

Conj-Her-1++:

The 6 mg/kg single-dose regimen resulted in the maximum possible tumorgrowth delays. There were ten survivors with a mean tumour volume of 161mm³. There were 5 partial regressions, 5 complete regressions and 2tumour free survivors.

(vii) NCI-N87

Conjugates tested: Conj-Her-1, Conj-Her1+, Conj-Her-1++

Female CB.17 SCID mice, aged eight weeks, were injected with 0.1 ml of1×10⁷ NCI-N87 in 50% Matrigel cells subcutaneously in the right flank.When tumours reached an average size of 100-150 mm³, treatment began.Mice were weighed twice a week. Tumour size was measured twice a week.Animals were monitored individually. The endpoint of the experiment wasa tumour volume of 800 mm³ or 83 days, whichever came first.

Groups of 10 xenografted mice were injected i.v. with 0.2 ml per 20 g ofbody weight of antibody drug conjugate (ADC) in phosphate bufferedsaline (vehicle) or with 0.2 ml per 20 g of body weight of vehiclealone.

The concentration of ADC was adjusted to give the following doses:

Conjugate Doses (mg ADC/kg body weight) Conj-Her-1  6, 18 Conj-Her-1+ 3,6 Conj-Her-1++ 1.5, 3  

All regimens were acceptably tolerated with little body weight loss.

The median time to end point (TTE) for vehicle-treated controls was 36.8days, establishing a maximum possible tumour growth delay (TGD) of 46.2days (126%) for the 80-day study.

Conj-Her-1:

The 6 and 18 mg/kg regimens resulted in tumor growth delays of 45.9 days(125%) and 46.2 days (126%). In the 6 mg/kg regimen the mean tumourvolume for 4 mice was 564 mm³, with no observable regression responses.In the 18 mg/kg regimen there were nine survivors with a mean tumourvolume of 108 mm³. There were 9 partial regressions and 1 completeregressions.

Conj-Her-1++:

The 1.5 and 3 mg/kg regimen resulted in tumor growth delays of 45.9 days(125%) and 46.2 days (126%). In the 1.5 mg/kg regimen there were twosurvivors with a mean tumour volume of 634 mm³. In the 3 mg/kg regimenthere were ten survivors with a mean tumour volume of 451 mm³.

Conj-Her-1+:

The 3 and 6 mg/kg regimens resulted in maximum tumor growth delays of46.2 days (126%). In the 3 mg/kg regimen there were seven survivors witha mean tumour volume of 600 mm³. In the 6 mg/kg regimen there were tensurvivors with a mean tumour volume of 256 mm³. There were two partialregressions.

(viii) JIMT1

Conjugates tested: Conj-Her-1, Conj-Her-1++

Female CB.17 SCID mice, aged eight weeks, were injected with 0.1 ml of1×10⁷ JIMT1 in 50% Matrigel cells subcutaneously in the right flank.When tumours reached an average size of 100-150 mm³, treatment began.Mice were weighed twice a week. Tumour size was measured twice a week.Animals were monitored individually. The endpoint of the experiment wasa tumour volume of 1000 mm³ or 60 days, whichever came first.

Groups of 10 xenografted mice were injected i.v. with 0.2 ml per 20 g ofbody weight of antibody drug conjugate (ADC) in phosphate bufferedsaline (vehicle) or with 0.2 ml per 20 g of body weight of vehiclealone.

The concentration of ADC was adjusted to give the following doses:

Conjugate Doses (mg ADC/kg body weight) Conj-Her-1 18, 24 Conj-Her-1++4, 6, 8

All regimens were acceptably tolerated with little body weight loss.

The median time to end point (TTE) for vehicle-treated controls was 37.5days, establishing a maximum possible tumour growth delay (TGD) of 22.5days (60%) for the 60-day study.

Conj-Her-1:

The 18 and 24 mg/kg regimens resulted in tumor growth delays of 5.3 days(14%) and 3.2 days (9%). In the 18 mg/kg regimen all animals reached the1000 mm³ endpoint. In the 24 mg/kg regimen there was a single survivorwith a tumour volume of 968 mm³. Both regimens resulted in a significantoverall survival difference versus controls (P<0.01).

Conj-Her-1++:

The 4, 6 and 8 mg/kg regimen resulted in tumor growth delays of 4.4 days(12%). 6.9 days (18%) and 17.7 days (47%). In the 4 mg/kg regimen allanimals reached the 1000 mm³ endpoint. In the 6 mg/kg regimen there wasa single survivor with a tumour volume of 650 mm³. In the 8 mg/kgregimen there was a single survivor with a tumour volume of 847 mm³. Allregimens resulted in a significant overall survival difference versuscontrols (P<0.01).

Example 9—Toxicology Assay

A single dose nonGLP toxicity study was used to determine the maximumtolerated dose (MTD) and safety profile of the ADCs tested, which were:Conj-R347-1; Conj-R347-2; Conj-R347-3.

Male Sprague Dawley rats (Envigo, Inc) were dosed once by slow bolusintravenous injection via the tail vein with ADC. The vehicle fordilution used was 25 mM Histidine-HCl, 7% sucrose, 0.02% Polysorbate 80,pH 6.0. Parameters evaluated during the study included mortality,physical examinations, cageside observations, body weights, body weightchanges, clinical pathology (clinical chemistry, hematology, andcoagulation), and gross pathology findings. All animals were terminatedon Study Day (SD) 29.

Conj-R347-1

Group Dose (mg/kg) N 2 5 5 4 7 5 6 10 5 8 16 5 10 20 5 12 25 5

Tolerability was determined based on toxicity end points, including milddecreases in hematological parameters, microscopic evaluations and bonemarrow suppression. Based on microscopic changes in animals receivingthe highest dose, the maximum tolerated dose (MTD) in the rat after asingle dose was determined to be 25 mg/kg.

Conj-R347-2

Group Dose(mg/kg) N 2 2 5 3 5 5 4 8 5

Tolerability was determined based on toxicity end points. The ADC wastolerated up to 8 mg/kg in the rat after a single dose. Findingsincluded dose dependent body weight decrease and bone marrowsuppression.

Conj-R347-3

Group Dose (mg/kg) N 5 1 5 9 2.5 5 11 4 5

Tolerability was determined based on toxicity end points. The ADC wastolerated up to 4 mg/kg in the rat after a single dose. Findingsincluded dose-dependent body weight loss and bone marrow suppression.

Therapeutic Index

The therapeutic index of each ADC/drug linker was calculated using thefollowing equation:

TI=MTD in rat (mg/kg)/MED in mouse efficacy model (mg/kg)

NCI-N87 Drug Linker MTD (mg/kg) MED (mg/kg) TI 1 25 6 4.2 2 8 2 4 3 4 14

1.-66. (canceled)
 67. A compound of formula I:

and salts and solvates thereof, wherein: R⁶ and R⁹ are independentlyselected from H, R, OH, OR, SH, SR, NH₂, NHR, NRR′, nitro, Me₃Sn andhalo; where R and R′ are independently selected from optionallysubstituted C₁₋₁₂ alkyl, C₃₋₂₀ heterocyclyl and C₅₋₂₀ aryl groups; R⁷ isselected from H, R, OH, OR, SH, SR, NH₂, NHR, NRR′, nitro, Me₃Sn andhalo; R″ is a C₃₋₁₂ alkylene group, which chain may be interrupted byone or more heteroatoms, e.g. O, S, NR^(N2) (where R^(N2) is H or C₁₋₄alkyl), and/or aromatic rings, e.g. benzene or pyridine; Y and Y′ areselected from O, S, or NH; R^(6′), R^(7′), R^(9′) are selected from thesame groups as R⁶, R⁷ and R⁹ respectively; R^(11b) is selected from OH,OR^(A), where R^(A) is C₁₋₄ alkyl; and R^(L) is a linker for connectionto a cell binding agent, which is selected from: (iiia):

wherein Q is:

where Q^(X) is such that Q is an amino-acid residue, a dipeptide residueor a tripeptide residue; X is:

where a=0 to 5, b=0 to 16, c=0 or 1, d=0 to 5; G^(L) is a linker forconnecting to a Ligand Unit; and (iiib):

where R^(L1) and R^(L2) are independently selected from H and methyl, ortogether with the carbon atom to which they are bound form acyclopropylene or cyclobutylene group; and e is 0 or 1; either (a) R²⁰is H, and R²¹ is OH or OR^(A), where R^(A) is C₁₋₄ alkyl; or (b) R²⁰ andR²¹ form a nitrogen-carbon double bond between the nitrogen and carbonatoms to which they are bound; or (c) R²⁰ is H and R²¹ is SO_(z)M, wherez is 2 or 3 and M is a monovalent pharmaceutically acceptable cation; or(d) R²⁰ is H and R²¹ is H; or (e) R²¹ is OH or OR^(A), where R^(A) isC₁₋₄ alkyl and R²⁰ is selected from: (d-i)

(d-ii);

(d-iii)

 where R^(Z) is selected from: (z-i)

(z-ii) OC(═O)CH₃; (z-iii) NO₂; (z-iv) OMe; (z-v) glucoronide; (z-vi)—C(═O)—X₁—NHC(═O)X₂—NH—R^(ZC), where —C(═O)—X₁—NH— and —C(═O)—X₂—NH—represent natural amino acid residues and R^(ZC) is selected from Me,OMe, OCH₂CH₂OMe.
 68. The compound according to claim 67, wherein both Yand Y′ are O and R″ is: (a) C₃₋₇ alkylene; or (b) a group of formula:

where r is 1 or
 2. 69. The compound according to claim 67, wherein R⁹ isH and R⁶ is H.
 70. The compound according to claim 67, wherein R⁷ is aC₁₋₄ alkyloxy group.
 71. The compound according to claim 67, whereinR^(6′) is the same group as R⁶, R^(7′) is the same group as R⁷, R^(9′)is the same group as R⁹ and Y′ is the same group as Y.
 72. The compoundaccording to claim 67, wherein R²⁰ and R²¹ form a nitrogen-carbon doublebond between the nitrogen and carbon atoms to which they are bound. 73.The compound according to claim 67, which is of formula Ia, Ib or Ic:

where R^(1a) is selected from methyl and benzyl; R^(L) and R^(11b) areas defined in claim
 67. 74. The compound according to claim 67, whereinR^(11b) is OH.
 75. The A compound according to claim 67, wherein R^(L)is of formula IIIa, and Q is a dipeptide residue selected from:^(CO)-Phe-Lys-^(NH), ^(CO)-Val-Ala-^(NH), ^(CO)-Val-Lys-^(NH),^(CO)-Ala-Lys-^(NH), ^(CO)-Val-Cit-^(NH), ^(CO)-Phe-Cit-^(NH),^(CO)-Leu-Cit-^(NH), ^(CO)-Ile-Cit-^(NH), ^(CO)-Phe-Arg-^(NH), and^(CO)-Trp-Cit-^(NH).
 76. The compound according to claim 67, whereinR^(L) is of formula IIIa and a is
 0. 77. The compound according to claim67, wherein R^(L) is of formula IIIa and b is 0 to
 8. 78. The compoundaccording to claim 67, wherein R^(L) is of formula IIIa and d is
 2. 79.The compound according to claim 67, wherein R^(L) is of formula IIIa andG^(L) is selected from:

where Ar represents a C₅₋₆ arylene group.
 80. The compound according toclaim 79, wherein G^(L) is G^(L1-1).
 81. The compound according to claim67, wherein the compound is of formula Id:

where Q is selected from: (a) —CH₂—; (b) —C₃H₆—; and (c)


82. A conjugate of formula II:L-(D^(L))_(p)  (II) wherein L is a Ligand unit, D^(L) is a Drug Linkerunit of formula I′:

wherein R⁶, R⁷, R⁹, R^(11b), Y, R″, Y′, R^(6′), R⁷, R^(9′), R²⁰ and R²¹,are as defined in any one of claims 1 to 8; R^(LL) is a linker forconnection to a cell binding agent, which is selected from: (iiia):

where Q and X are as defined in claim 67 and G^(LL) is a linkerconnected to a Ligand Unit; and (iiib):

where R^(L1) and R^(L2) are as defined in claim 67; wherein p is aninteger of from 1 to
 20. 83. The conjugate according to claim 82,wherein G^(LL) is selected from:

where Ar represents a C₅₋₆ arylene group.
 84. The conjugate according toclaim 83, wherein G^(LL) is G^(LL1-1).
 85. The conjugate according toclaim 82, wherein D^(L) is of formula (Id′):

where Q is selected from: (a) —CH₂—; (b) —C₃H₆—; and (c)


86. The conjugate according to claim 82, wherein the Ligand Unit is anantibody or an active fragment thereof which binds to one or moretumor-associated antigens or cell-surface receptors selected from(1)-(88): (1) BMPR1B; (2) E16; (3) STEAP1; (4) 0772P; (5) MPF; (6)Napi3b; (7) Sema 5b; (8) PSCA hIg; (9) ETBR; (10) MSG783; (11) STEAP2;(12) TrpM4; (13) CRIPTO; (14) CD21; (15) CD79b; (16) FcRH2; (17) HER2;(18) NCA; (19) MDP; (20) IL20R-alpha; (21) Brevican; (22) EphB2R; (23)ASLG659; (24) PSCA; (25) GEDA; (26) BAFF-R; (27) CD22; (28) CD79a; (29)CXCR5; (30) HLA-DOB; (31) P2X5; (32) CD72; (33) LY64; (34) FcRH1; (35)IRTA2; (36) TENB2; (37) PSMA—FOLH1; (38) SST; (38.1) SSTR2; (38.2)SSTR5; (38.3) SSTR1; (38.4) SSTR3; (38.5) SSTR4; (39) ITGAV; (40) ITGB6;(41) CEACAM5; (42) MET; (43) MUC1; (44) CA9; (45) EGFRvIII; (46) CD33;(47) CD19; (48) IL2RA; (49) AXL; (50) CD30—TNFRSF8; (51) BCMA—TNFRSF17;(52) CT Ags—CTA; (53) CD174 (Lewis Y)—FUT3; (54) CLEC14A; (55)GRP78—HSPA5; (56) CD70; (57) Stem Cell specific antigens; (58) ASG-5;(59) ENPP3; (60) PRR4; (61) GCC—GUCY2C; (62) Liv-1—SLC39A6; (63) 5T4;(64) CD56—NCMA1; (65) CanAg; (66) FOLR1; (67) GPNMB; (68) TIM-1—HAVCR1;(69) RG-1/Prostate tumor target Mindin—Mindin/RG-1; (70) B7-H4—VTCN1;(71) PTK7; (72) CD37; (73) CD138—SDC1; (74) CD74; (75) Claudins—CLs;(76) EGFR; (77) Her3; (78) RON—MST1R; (79) EPHA2; (80) CD20—MS4A1; (81)Tenascin C—TNC; (82) FAP; (83) DKK-1; (84) CD52; (85) CS1—SLAMF7; (86)Endoglin—ENG; (87) Annexin A1—ANXA1; (88) V-CAM (CD106)—VCAM1.
 87. Theconjugate according to claim 82 wherein p is an integer from 1 to
 8. 88.A pharmaceutical composition comprising the conjugate of claim 82, and apharmaceutically acceptable diluent, carrier or excipient.
 89. A methodof treating a mammal having a proliferative disease, comprisingadministering to the mammal an effective amount of a conjugate accordingto claim 82.